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Building Synthetic Cells─From the Technology Infrastructure to Cellular Entities
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ACS Synthetic Biology

Cite this: ACS Synth. Biol. 2024, 13, 4, 974–997
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https://doi.org/10.1021/acssynbio.3c00724
Published March 26, 2024

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Abstract

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The de novo construction of a living organism is a compelling vision. Despite the astonishing technologies developed to modify living cells, building a functioning cell “from scratch” has yet to be accomplished. The pursuit of this goal alone has─and will─yield scientific insights affecting fields as diverse as cell biology, biotechnology, medicine, and astrobiology. Multiple approaches have aimed to create biochemical systems manifesting common characteristics of life, such as compartmentalization, metabolism, and replication and the derived features, evolution, responsiveness to stimuli, and directed movement. Significant achievements in synthesizing each of these criteria have been made, individually and in limited combinations. Here, we review these efforts, distinguish different approaches, and highlight bottlenecks in the current research. We look ahead at what work remains to be accomplished and propose a “roadmap” with key milestones to achieve the vision of building cells from molecular parts.

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Introduction: What Is a Cell and Why Build One?

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What Is a Cell?

Cells are discrete, compartmentalized units of living systems that are distinct from their surrounding environment and other cells. As individuals, they can interact with each other, the environment, and act as distinct units of selection in evolution. While most living cells comprise lipid-bounded compartments, other biological entities, such as complex viruses, rely on protein capsules. Growing evidence suggests that compartmentalization via liquid–liquid phase separation (i.e., coacervate formation) may also be sufficient for compartmentalization. (1)
The flexibility of this definition of a cell begs the question, “What is life?” While this has been debated for decades, if not centuries, even a modern, more complete understanding of biological systems at the molecular level has not yielded a consensus definition. (2) This is for good reason. Earth’s organisms (the only ones known so far) demonstrate breathtaking diversity in their ecology, phenotypes, and biochemistry. Definitions of life have tended to search for commonality in life, and thus are repeatedly rewritten, following discoveries that disprove previously established rules. (3,4) Perhaps a generalized universal definition of life is even impossible: a “natural kind” in philosophy is a category that reflects the actual world and not just human interests or properties of a group. (5) For example, water is a natural kind, whereas chairs are not. Life is possibly not a natural kind, and thus there may never be a natural definition. (6)
To anchor our discussion, we use Gánti’s Chemoton model of life, (7) which uses three criteria: replication, metabolism, and compartmentalization. From these criteria emerge additional features, such as evolution, responsiveness to stimuli, and directed movement, which are strongly indicative but not strictly required for life. (8) Although the latter three emerge from the three main criteria, they are not strictly limited to “fully living” systems. For example, a replicating moving system need not necessarily show compartmentalization. The primary criteria are interdependent. For example, replication cannot occur without the compartmentalization of “self” genetic material from “non-self” genetic material. The merits of these three essential criteria have been discussed at length elsewhere. (9−11,6) Gray areas are immediately apparent. What about “replication” through mechanical processes? Metabolism for how long? Evolution is a derived characteristic of populations, not individuals, and not all cells have the ability for directed movement and responsiveness to stimuli. Ultimately, the key question is if we were to engineer a living system de novo, what are the desired final parameters by which we declare success? This process itself may force another re-examination of what it means to be “alive”.
For simplicity, we propose that any engineered synthetic system that meets the three criteria, and subsequently demonstrates evolution and possibly other emergent behaviors, should qualify as “alive”, as depicted in Figure 1. The word “system” is key, as life is a population-level phenomenon. For example, not every member must replicate, and certainly no individual undergoes evolution. This definition also works well with other working definitions of life. The so-called NASA definition of life, “a self-sustaining chemical system capable of Darwinian evolution” (9,12) includes the critical phrase “self-sustaining”, which we include in our discussion. Note that in this context “self-sustaining” does not mean completely autotrophic, as all organisms require the intake of water and elements, such as carbon, nitrogen, and other nutrients. Thus, this includes everything from diazotrophic cyanobacteria to parasites. In reality, “self-sustaining” refers to being able to live without outside intervention. Probably a better way to put it is that, while alive, an organism sustains itself in the face of entropy. We expect that as more discoveries are made, especially if life is discovered beyond Earth, the three criteria may continue to evolve, potentially allowing us to formulate a natural definition of life.

Figure 1

Figure 1. Our working definition of living systems, based on Tibor Gánti’s Chemoton model of life. Compartmentalization, replication, and metabolism together make up minimal criteria for a living system. From these other features emerge, including evolution, responsiveness to stimuli, and directed movement.

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Here, we discuss the varying strategies and approaches to build synthetic cells. We then describe the motivations for and impact of building synthetic cells by highlighting the features of life that make biology attractive as a technology. Finally, we identify the progress and most critical current and future challenges in the field. This leads us to propose a roadmap toward the design and synthesis of a wholly engineered living system that embodies the criteria of life as put forward here. We envision this endeavor to be accomplished within a decade.

Why Build a Cell?

What is to be gained from building a cell? While the breadth of life on Earth is awe-inspiring in its diversity, the exercise of building a cell de novo offers lessons and perspectives that are unique from the scientific interrogation of extant living systems: it could enable entirely new science and may empower the engineering of living systems that are orthogonal and contextually superior to those that have emerged naturally. Examples of scientific gains from this pursuit include insights into how life on Earth may have begun, as well as what may permit life’s emergence elsewhere in the universe. Practical applications could include the creation of better platforms for industrial biotechnology and biomedicine.
The tools for building cells and dissecting the function of biological mechanisms into modules will entail bioengineering advances that affect multiple areas of the life sciences, (13) if the development of tools for the experimental modification of living systems is a guide. Such experiments interrogate cellular systems by endowing them with novel functions and expanded capabilities. While similar to metabolic engineering efforts, such gain-of-function experiments are distinct in that they primarily aim to answer questions about the system and seek to replicate characteristics of life from nonliving components to shed light on fundamental questions about life’s origins, components, and workings. (14,15)
Building a cell would likely have profound scientific ramifications: a fundamental understanding of all cellular components and their organization required to build a living cell would significantly advance our ability to comprehend how the highly complex, dynamic, interacting system called “life” operates. Equally important, the ability to build cells would expand the range of cell-types beyond the one shared by all life on Earth, thus giving scientists the ability to study not just life as it is, but as it could be. Currently, we have one example of a successful cellular system: that from which all life on Earth descends. Surely, there must be other feasible compositions of functional cellular systems. Others may well have existed on Earth, but we have yet to recognize any traces they left─alternatives may also exist in places beyond Earth, but also those have yet to be discovered.
The first synthetic cells will likely be very basic and thus extremely sensitive to environmental changes. They would be “minimal” as compared to the functions of common model microbes, such as Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae, which have evolved naturally and are thus robust in respect to a wide range of environmental conditions. It will likely require the infrastructure of generations of professionals with the enabling technologies to build, tune, and enhance or optimize features of many different synthetic cells for particular applications. For instance, a synthetic cell optimized for industrial biotechnology, such as the production of chemicals, is unlikely to also be optimized for therapeutic uses, such as killing cancer cells. Thus, the greatest values of cells created de novo will not be represented in the functions of the first iterations of a synthetic cell, but in the opportunities it opens up.

Advantages of Synthetic Cells over Cell-Free or Liposome Systems

Why are existing technologies with widespread commercial availability, such as cell-free transcription/translation (Tx/Tl) systems, not sufficient? What are the limitations of encapsulated cell-free systems in studying life? To answer this, we have distinguished four principal systems as shown diagrammatically in Figure 2, while their advantages and disadvantages are summarized in Table 1.

Figure 2

Figure 2. Four different molecular platforms for studying life. Outgoing from externally provided template DNA (1), a cell-free transcription/translation (Tx/Tl) system uses complex substrates and a chemical energy-donor (2) to synthesize RNA, proteins and potentially other biocatalytically (enzymatically) formed products (3). An encapsulated cell-free system is often a liposome enclosing a cell-free Tx/Tl system. A synthetic cell would function more like a natural cell in that it could be a self-sustaining system that is capable of replication.

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Table 1. Advantages and Disadvantages of Using Different Molecular Platforms for Studying Life
 cell-freeencapsulated cell-freesynthetic cellnaturally evolved cell
Defining life/Origin-of-lifeHelps understand certain processesHelps understand a broader range of processesIdeal to understand the transition from nonliving to livingNoncontroversial example of life
Alternative cellular systemsMost easily modified for noncanonical componentsNoncanonical components functioning in a unitNo constraints of prior evolution, genetics and developmentChanges are constrained by evolution, genetics and development
Studying genes of unknown functionTesting of genes against a defined backgroundTesting of genes against a defined backgroundTesting of genes against a defined backgroundToo many to study; throughput of traditional means too low and slow; reached limit of previous approaches, better to use synthetic biology tools
Prototyping metabolic pathwaysIdeal for simple pathways; no concern about lethality to a cellEnables compartmentalization for more sophisticated pathwaysMost orthogonal while sufficiently capable for more complex pathwaysLeast orthogonal but most capable for complex pathways
Molecular biosynthesis developmentTesting of metabolite transformationTesting expansion of metabolite transformationIntegrated multigene pathway testing in controlled/known environmentAnalysis of alternative/competing molecular pathways
Production of toxic compoundsIdeal/unrestrictedBiocontained delivery of biotechIdeal if not toxic to synthetic cell; potential to study mechanism of toxicityProhibited by definition
Preservation of biodiversityUnderstand key metabolic pathways with ecological importance, (e.g., plant–microbe interactions)Study and engineer key evolutionary and ecological processes (e.g., symbiogenesis)Study and engineer symbiogenesis, preserve extant and extinct life (e.g., boot-up endangered or extinct genomes), generate new biodiversityPreserve organism with intrinsic value and natural producer of valuable compound
Designing environmental stress responsesProtein stability testingBasic functional tests (e.g., protection of DNA from radiation), subcellular localization and compartmentalization, transport phenomenaResponse testing in controlled/known environmentInteraction of stress response with other cellular mechanisms
Therapeutics production/delivery/testingDistributed production, logic-gated controlCompartmentalization and targeted delivery, logic-gated controlAllow testing against a known genetic background; could provide orthogonal system for drug deliveryMay have superior compatibility with life
Studying cellular functionDependent on system used for Tx/Tl“Toy” system amenable to modeling; reintroducing confinement/organelles/scaffolding in controlled wayProvides modular, defined systemIntact systems for realistic studies
Evolution studiesComponent evolution (e.g., genes)Component evolution (e.g., transport proteins)Studies with well-defined cellular systemsNatural evolutionary processes
Workflows for engineered biological “parts” (modules)Testing parts in a simplified system toward use in living cellsTesting parts in a simplified system toward use in living cells, with more realistic spatial confinement/biophysics than bulk cell-freeTesting of parts against a defined backgroundDifficult/impossible/unmet challenge to predict a priori how engineered part will function in vivo
The key difference between a cell-free system and a cell is compartmentalization. This allows for the emergence of individuality in metabolism, heredity, and evolution. Thus, a synthetic cell could be the superior tailored platform for basic science and bioengineering. Like naturally evolved life, a synthetic cell may survive in a suspended state for longer periods of time than typical cell-free systems, which is critical when preservation of cellular components is key. Additionally, a synthetic cell could provide a unit of selection for the evolution of desired processes, whereas a cell-free system is not capable of that by a homogenized whole.

Is There an Ideal Synthetic Cell?

What are the ideal properties of a synthetic cell? Biologists and engineers dream of an organism in which every chemical interaction is defined and understood so that the phenotype can be predicted from the genotype. The resulting genetic transparency is somewhat congruent with the idea of a “minimal cell”. Such an organism would serve as a genetic “base operating system” onto which any desired function can be “installed”. In further analogy to computer science, difficulties in the engineering of natural cells arise from their “closed-source” programming and the difficulty to reverse-engineer them and accurately back-translate their compiled “machine-code”. In contrast, synthetic cells would be “open access” making the “source-code” available and thereby allowing the more precise “programming” of functions before compilation.
Transitioning from the theoretical attributes of a synthetic cell to a chemical understanding and implementation may be difficult. Because life is an emergent property of chemistry, it is by definition stochastic and unpredictable. (16) On another level that accounts more for context, engineers tend to have specific, application-dependent needs, relating to growth-rate and biomass-yield, source of energy, carbon, and other nutrients, as well as robustness within an environment (e.g., temperature and pH) and ecology. These are determined by the intended purpose of the organism (foundational studies, metabolic engineering, biomedicine, etc.). In short, there is no single “ideal” synthetic cell, as the design depends on the need and intended application/use case. Thus, not all synthetic cells will necessarily be the most minimal.

Strategies for Building a Cell

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The ultimate goal of building a cell is to create a biochemical system recognizable as alive from its nonliving component parts de novo. Different design strategies for building a cell offer relevant approaches to provide platforms for the development of foundational technologies and advancing understanding of cellular structures and functions toward the de novo creation of a cell. These alternative approaches can be organized into “bottom-up”, “top-down”, and “middle-out”. Each approach is characterized by unique advantages, drawbacks, and challenges. A schematic overview of all three strategies is represented in Figure 3.

Figure 3

Figure 3. Major approaches to building a synthetic cell: bottom-up (a), top-down (b), and middle-out (c) are distinguished as the basic concepts of currently ongoing research. Top-down approaches generally seek to find a “minimal genome” of an existing organism, while the bottom-up approach aspires to de novo create a cell “from scratch” or based on macromolecules. Middle-out approaches utilize modules of known function (e.g., organelles, extracts) to assemble a new cell or major elements/mechanisms thereof.

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To help illustrate these different approaches, consider the analogy of building a car that can drive. The bottom-up approach is like building a drivable car from the most basic components, such as metal sheeting and screws. The top-down approach is akin to stripping a drivable car piece-by-piece, discarding “non-essential” parts, modules, and subsystems (e.g., the radio) while retaining the ability to drive. Sometimes, which modules are “non-essential” is unclear until the car stops driving. The middle-out approach is akin to putting a car together from subsystems and modules (e.g., the engine) whose intricacies the assembling engineer does not need to fully comprehend. However, unlike a car, a living cell displays emergent properties that are extremely difficult to predict and are profound in their implications.
In the following section, we briefly introduce the different approaches and outline the efforts to-date that have been undertaken on each. We also compare the merits of the different approaches and discuss the scientific and technical challenges.

Bottom-up Engineering

The bottom-up approach comprises the step-by-step chemical synthesis of a living system de novo, from isolated or synthesized (macro)molecules that can comprise membranes, genetic material, and proteins to carry out the functions of a cell. This gives the researcher complete control of the components, whether to try to create an orthogonal system with nonbiological components, such as D-amino acids and L-sugars, or to try to replicate the origin-of-life here or elsewhere. This approach operates on the hypothesis that the right configuration of molecules will display life-like behaviors, or ultimately create a system we recognize as “life”. It arguably best exemplifies the ultimate goal of the field, but may require the greatest depth of knowledge and technical skill to be accomplished.
There has been much work toward bottom-up engineering to understand the origin-of-life, creating liposomes that encapsulate various portions of the genetic machinery. (17−19) Polymersomes, artificial vesicles that self-assemble from amphiphilic copolymers and enclose an aqueous cavity, (20) and synthosomes, polymersomes that contain channels to allow for selective transport of chemicals, are more recent advances that stray further from mimicking natural organisms. Polymersomes can be engineered as protocells to mimic cell functions and build cell-like structures. (21) However, there is still a substantial gap between the abiotic production of macromolecules, or even macromolecular assemblages, such as, vesicles loaded with biochemicals, and living systems. Also, as origin-of-life research specifically seeks to recreate life as it likely occurred on a young Earth, the field often does not explore radically different alternatives that might interest an astrobiologist, synthetic biologist, or biomolecular engineer.

Top-down Engineering

Systematic top-down approaches commonly aim to remove extraneous genetic modules from extant genomes to generate a “minimal genome” that contains a subset of its initial functions. (22,23) The objectives of these range from enabling a simplified platform for bioengineering to creating a cell with only the essential functions required to sustain cellular life for foundational studies. (24−26) The approach has been applied to various species and genetic systems to varying extents. For example, the genome of E. coli was reduced by up to 30%. (27,28) The most minimal genome was created from the bacterium Mycoplasma mycoides, whose genome is already one of the smallest known with only 915 genes. From this species, the synthetic genome JCVI-syn1.0 was created by developing a series of enabling synthetic biology technologies. (24,25,29−32) There, a synthetic bacterial genome containing an antibiotic resistance marker was installed in an existing living bacterial cell to produce a new cell with the genotype of the installed genome. Consequently, a near-minimal organism was created with a synthetic genome that comprised only 531 kbp and 473 genes, a genome smaller than that of any autonomously replicating cell found in nature. While several species exist that have fewer genes than Mycoplasma genitalium, none of them are free-living. Notably, the progenitor, JCVI-syn3.0, can only be cultivated in a laboratory environment under strictly controlled conditions. (26)

Middle-out Engineering

The middle-out or “semi-synthetic” approach extracts and repurposes extant modules of living cells to reconstruct a living system. (33) Much of the research now designated as a middle-out approach was previously considered bottom-up. The classic example is the use of complex fractions of cell extracts, such as organelles and other modularly functional fractions of living material that enable functional biochemistry, to reconstitute features of living systems, such as gene expression, replication, and sense/response behavior. (34) Examples include the encapsulation of cell-extracts in liposomes to achieve cellular functions, (35,36) decorating nanoparticles with cell membranes, (37) and re-engineering and transferring complex molecular systems, such as the glycosylation machinery, to synthetic systems, such as a microsome. (38) Synthetic biologists have recreated fairly complex systems of replication and transcription-coupled translation in vitro. (39,40) Encapsulating such systems into membranes and coupling their function to cytokinesis and metabolism is still a profound challenge. (34)
The middle-out approach is distinct from the top-down approach in that it does not rely on the minimization of naturally evolved cells. It is distinct from the bottom-up approach in that it does not rely on purified or synthesized, well-characterized molecules with defined functions but instead utilizes only partially understood multimolecular assemblies to perform such functions. Such middle-out systems can be treated as modular black boxes with defined inputs and outputs. Despite being incompletely understood and some lack of reproducibility, these systems can still be leveraged to achieve life-like behavior and are nonetheless useful in piecing together the essential features of living systems, either in conjunction with other complex mixtures or purified molecules. In fact, the reconstitution of a living cell from its components following extraction has yet to be accomplished and would provide a worthy milestone as a technical achievement for the insight it could give into what differentiates living entities from nonliving systems.

Applications of Synthetic Cells

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Synthetic cells are anticipated to have a range of applications, from fundamental research to applied science. For bioprocess engineers, synthetic cells are of interest as an infinitely scalable manufacturing platform for chemicals or for environmental applications. Synthetic cells could also offer an alternative platform for biomanufacturing that is not readily performed by existing cellular chassis. For example, synthetic cells could allow the unbiased implementation of nonevolved elements like nonstandard (proteogenic) amino acids (NSAAs), (41) alternate chiralities, and xenonucleic acids (XNAs) (42) that are inaccessible or hardly accessible with extant biology. Cell biologists would be able to use synthetic cells to understand the fundamental workings of living cellular systems. Evolutionary biologists and astrobiologists could use them to answer the “what if?” questions of the origin, evolution and diversity of life. For example, could life originate in multiple ways? What does that say about the potential for life elsewhere? The following examples are meant to illustrate the impact synthetic cells could have and thus the importance of the endeavor to build a cell.

Understanding the Origin, Evolution, and Diversity of Life

The physicist Richard Feynman famously wrote, “What I cannot create, I do not understand.” Building a cell may thus be the only way to truly provide a mechanism for the ultimate validation of our foundational understanding of life’s components and functions. Our inability to create a synthetic cell from molecular components─even from components derived from previously alive cells─illuminates gaps in our understanding.
Most likely, all extant life on Earth descended from a universal common ancestor. (43) The exercise of building a cell could allow us to postulate what life might have looked like elsewhere in our solar system and beyond, perhaps permitting us to answer one of our most profound scientific questions: are we alone? Further, synthetic cells could offer a platform for understanding how life might operate on specific extraterrestrial bodies, such as the subsurface oceans of Europa and Enceladus. In addition to “how”, we are left with many of the “why” questions of evolution. Why does life on Earth only use left-handed amino acids in ribosomally synthesized proteins? Why D-sugars? Did evolution sample other approaches and simply discard them out of pure chance? Or were they outcompeted? Or did alternatives never arise because none were functional? Building synthetic cells would allow us to distinguish among these alternatives for a basic scientific understanding of life. It can further provide insight into the biochemical and structural intricacies of cellular functions to guide application-focused bioengineers. Evidence that the molecules involved in the central dogma on Earth are not the only “way of life” is provided in the form of DNA with unnatural base pairs (UBPs), consisting of, e.g., six bases instead of four. (44) This DNA also seems to be energetically superior, as the two non-natural bases are less prone to oxidation and epimerization. The creation of an E. coli strain that actually uses unnatural base pairs shows the feasibility of such hypotheses. (45)

Characterization of Cellular Parts and Elucidation of their Functions

Synthetic cells could allow the testing of biological parts outside of their native context. Such “parts” may range in size and complexity from lipid composition to the genetic code to whole organelles. If a module is necessary and sufficient for a particular desired function, a synthetic cell with minimal complexity could provide an excellent chassis to evaluate how that module affects or is affected by basic cellular functions. This is because the synthetic cell chassis is by concept a defined background free from the constraints, complexity, and legacy of evolution with the confounding variables and added complexity found in natural cells─in brief, synthetic cells could be entirely orthogonal. Dissecting the cellular machinery of a natural cell through deletion is not always possible or preferable to adding components to a synthetic cell. Currently, there are considerable efforts and hypotheses to understand adaptation and regulatory processes involving domestication and biodiversity of natural organisms. (46) Synthetic cells may also be useful as foundations for the synthetic domestication of biodiversity traits─an approach that constitutes a sustainable and viable option for conservation and development of value-added processes and products from biodiversity. (47)

Development of Engineering and Design Tools

Just as cell-free Tx/Tl systems are used for the rapid prototyping of genetic circuits, synthetic cells will prove similarly useful for prototyping at higher levels of organization and cellular interactions, for example, between components or membrane-less compartments. Synthetic cells may offer a broad array of traits found in natural cells that can ultimately be combined in an “à la carte” format to engineer uniquely tailored cell systems. Consider how five Spiroplasma genes were added to a nonmotile organism, yielding a mutant of the minimized bacterium JCVI-syn3B capable of movement. (48) This is not dissimilar to how evolution has operated with bacteria, which have an extraordinary ability to exchange DNA through horizontal gene transfer, or for eukaryotes who have formed temporary symbioses or even incorporated symbionts that ultimately became heritable, such as mitochondria. (49) Progress toward engineering synthetic cells also involves developing orthogonal biochemistries. Examples include NSAAs, XNAs, (50) and an expanded genetic code with UBPs, (51) as well as de novo designed proteins, potentially enabling the formation of proteins with new structures and novel functions. (52)

Metabolic Engineering and Biomanufacturing

The ability to predict, design, and construct reliable and robust biochemical pathways is key to efficient metabolic engineering. Systems Biology, quantitative synthetic biology and integrated multiomics techniques (i.e., genomics, transcriptomics, proteomics, and metabolomics) grant insights that can significantly accelerate the development of microbial cell factories. (53−55) At present, improving synthetic cells for biomanufacturing is not achieved by strain engineering (because synthetic cells don't have engineerable strains yet) but instead by better understanding how cells work and how to build them de novo. The concept, known as systems metabolic engineering, reduces the Design-Build-Test-Learn (DBTL) cycles required to reach a strain that performs as desired and enables the rational creation of microbial cell factories. Nevertheless, it is still not (yet) possible to exactly predict outcomes of genetic interventions in even the best-studied model organisms. Already, cell-free systems have been shown to accelerate design cycles. (56,57) A fully defined synthetic cell may go beyond such approaches to provide a predictive platform, allowing bioengineers to further streamline systems metabolic engineering strategies. This may not only improve control over the process parameters (e.g., rate, titer, yield), (58) but also improve the precision and efficiency of biomanufacturing. (59) Minimization of crosstalk between peripheral and intersecting heterologous metabolic functions may allow formerly inaccessible biosynthetic pathways, for example, of complex natural products, to be (re)constructed. (60−62) The semisynthetic near minimal JCVI-syn3A (and derivative strains) is already being used as a chassis to evaluate the effects of adding new metabolic pathways on basic cellular biology. Its extreme metabolic simplicity makes it useful for such purposes; however, synthetic or semisynthetic cells that are similar to common model organisms could be much more useful than current minimal cells.
A fully defined biomanufacturing system tailored to its purpose may also allow for better process control through enhanced biosafety, as compared to natural cell systems: requirements for containment may be relaxed due to a lower chance of survival, in case of unintentional release. Intrinsic biological controls could be considered, such as “kill switches” after a certain number of generations or in response to a particular environmental change. (63,64) Alternatively, or additionally, a synthetic cell could be designed to be incompatible with natural life, therefore unable to interact with or contaminate it (for example, by using D- rather than L-amino acids, L-sugars instead of D-sugars, or UBPs with XNAs and/or NSAAs). Vice versa, synthetic systems that are different enough from nature may not be susceptible to “intruders”, such as bacterial- or phage-contamination. Such microbial chassis are already in development and could provide a firewall to prevent any exchange between the recoded organism and natural ecosystems. A recent example is an E. coli strain with an alternative codon usage where artificial tRNAs recognize different amino acids. (65)

Food Production and Bioremediation

Microbial biotechnology will play a crucial role in advancing food-production systems (e.g., agriculture, livestock) toward greater efficiency and sustainability. (66) Because existing genetically modified organisms cannot persist indefinitely and are often outcompeted by natural systems in a short period of time, synthetic cells could become a viable tool to, for example, replace chemical fertilizers (67) or tailor microbiomes of plants (68) and animals. (69,70) Similarly as for biomanufacturing/metabolic engineering applications, orthogonality and intrinsic safeguards of the synthetic cells could ensure the biosafety of the synthetic system.
Natural cells have been used successfully in environmental remediation efforts, such as treating and recovering concentrated nutrients, sequestering heavy metals, and decomposing plastics. (71−74) Synthetic cells may provide opportunities to improve and expand these efforts, when engineered to survive environments that preclude naturally evolved life. For example, synthetic cells could be created to be impervious to compounds that are toxic to life. Natural cells excel at processing natural compounds, but synthetic cells may fill an important niche in bioremediation by being tailored to function with and act on synthetic compounds.
Synthetic cells could also become field-deployable detectors for environmental monitoring to detect contaminants, such as pathogens or heavy metals. (75) These “biosensors” through their portable sizes and scalability could provide a warning system in hazardous situations where there is a risk of exposure to harmful levels of toxic chemicals or pathogens.

Diagnostics and Therapeutics

Our understanding of the molecular biology of cells, though incomplete, underpins much of modern medicine, shaping our comprehension of disease and pathogenicity mechanisms and influencing the development of diagnostics and therapeutics. Several diagnostic procedures rely on identifying cellular components or products, while many therapeutics act by influencing cells, cell products, or altogether replacing cells and tissues. It is clear how diseases and their treatments often lead back to the cell. In the words of the physician-scientist Rudolf Virchow, “no matter how we twist and turn, we shall eventually come back to the cell. Every pathological disturbance, every therapeutic effect, finds its ultimate explanation only when it is possible to designate the specific living cellular elements involved.” (76,77) Similar to the way molecular biology contributed to modern medicine, one can expect insights from synthetic cell endeavors to further advance medical practice, improving our understanding of disease pathologies, and informing novel diagnostics and therapeutic strategies.
Synthetic cell research may enable the development of novel biological functions that do not currently exist in nature but may be relevant for the treatment of different disease conditions. Today, natural cells form the basis of cellular therapies, spanning multiple therapeutic areas including regenerative medicine, immunotherapy, and cancer. There are 29 FDA-approved cell- and gene therapies with others at various stages of clinical development. (78) However, there have been major limitations with the manufacturing of natural cell-based therapeutics with variability in product specifications as well as other sourcing, supply chain, and storage challenges. (79) For instance, CAR T-cell therapy, while promising, is restricted by these limitations, leading to extensive duration and high costs. (80,81) Synthetic cells that are able to mimic the functions of these natural cells could significantly improve access to these therapies, allowing better supply, storage, and manufacturing at scale. (82) Toward that objective, droplet-based microfluidics techniques for producing synthetic cells are very promising, as they can combine complex designs (e.g., compartmentalization) with high consistency and large-scale production. (83)
Extracellular vesicles have also become an active area of research toward therapeutic applications. Limitations of these vesicles are the difficulty sourcing them and their variability, which limits the ability to control their quality. Current synthetic cell research is already tackling these challenges with the bottom-up assembly of synthetic vesicles for wound healing, or regenerative therapies. (84) Further synthetic cell research may lead to the development of synthetic vesicles for other disease conditions for which natural extracellular vesicles have demonstrated effect in preclinical studies.
The potential of synthetic transcription and translation systems to provide a greater shelf life and stability, by being freeze-dried and later rehydrated, has already been recognized: such medicines and vaccines could mitigate regional healthcare disparities, overcome geographical and environmental barriers, and improve global healthcare access. (85,86) Better yet would be the ability to create a synthetic cell system that is amenable to long-term desiccation, to address the problem of maintaining stem cells or other cellular-based manufacturing systems in an active or even semiactive form.
Another promising potential application of synthetic cells are specialized medicines and therapies. The rapid advancement of synthetic biotechnologies and research techniques has increased the accessibility, reliability, and prevalence of personalized medicine, resulting in treatments tailored to a patient’s unique needs, physiology, and genetics. (87) A personalized medicine regime may be vital for the successful treatment of disease effectively, while maintaining quality of life.
With tissues and organs being multicellular organizations of cells, another potential area of impact for synthetic cell research is organ and tissue transplantation. Terminal irreversible damage of the kidneys, lungs, heart, liver, etc. are treated with organ transplantation. However, there are limitations in the supply of these organs to patients. New synthetic tissue approaches based on synthetic cell research may one day provide alternative organ sources for transplantation.

Bioprinting

Bioprinting is the use of 3D-printing technology with materials that incorporate viable living cells as a component of bioinks to create structures–in essence, biological additive manufacturing. To date, the focus has primarily been on tissue and organ regeneration, (88) but other uses such as printing of plant or algal cells have been explored. (89) Challenges exist that potentially could be remedied by the use of synthetic cells as components of bioinks. (88) For example, they could be engineered to withstand conditions such as temperature or chemical composition of the carrier compound that would be difficult or impossible for naturally evolved cells to survive. There could be situations where cell division is not desired. A synthetic cell could be engineered to be more biocompatible with others or be made immune to infection from viruses and bacteria and remain undetected by a host immune system.

Space Exploration

Space exploration is limited by mass and volume constraints during launch, as much of the launch-mass is fuel. In the absence of resupply, storage and reliability issues are key. As life is self-replicating and synthetic biology can transfer the ability to convert available resources into a more tractable form (say, a single cell that could produce wood or rubber), biotechnology has the potential to be the key to human survival off-planet. (90,83) Cells, especially microbes, are exquisitely good at nanotechnology and comparatively low-maintenance. (91) The attainable savings in up-mass can be huge. (92) Creating synthetic cells that are better suited to off-planet environments than naturally evolved life could be the key to life-support and in situ resource utilization for manufacturing of items with regular demand on long-duration space missions, such as food, drugs, and materials. (93,94)

Research Advancements toward Fulfilling the Criteria of Life

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To refine a framework for bioengineers to understand current challenges in creating synthetic biological life, we review progress and begin to frame target specifications. For that, we adhere to the foregone criteria of life “compartmentalization, metabolism, and replication”, as well as the emergent features “evolution, responsiveness, and movement”. While we discuss each of these separately, in many cases research has also demonstrated their combination. For example, various synthetic compartmentalized systems already exhibit some form of metabolism and replication. (95−98)

Compartmentalization

Compartmentalization of cells into discrete units is required to distinguish life from the physical environment, from each other, such as oxidative and other cells-types (especially in multicellular organisms). From a biological point of view, physical separation allows distinct individuals to experience differential selection. From a physical point of view, compartmentalization allows a separation and concentration of cellular components and compounds in a manner that facilitates metabolism. Compartmentalization also allows for different metabolic activities to occur within the same cell that would otherwise interfere with each other, such as, e.g., oxidative phosphorylation and nitrogen fixation, (99) or β-oxidation. (100)
Internal compartmentalization exists in all domains of life. In prokaryotes, there are two major classes of organelles: those formed by a protein shell or lipid monolayer (e.g., lipid bodies, polyhydroxyalkanoate (PHA) granules, carboxysomes, magnetosomes, and gas vacuoles) or lipid bilayer. (101) Eukaryotic compartments in the form of membrane-bound organelles (e.g., nucleus, mitochondrion, chloroplast) are well-known. Additional compartmentalization occurs through biomolecular condensates formed by liquid–liquid phase separation. (102,103) Examples from eukaryotes include membrane-less organelles in the nucleus, such as the nucleolus, Cajal bodies, nuclear speckles, and paraspeckles, and in the cytoplasm, such as P-bodies, stress granules, and germ granules. (104) Some eukaryotic organelles, such as mitochondria and chloroplasts, arose via the assimilation of bacteria into eukaryotic cells through endosymbiosis. (105) Synthetic biology approaches have even enabled the establishment of a bacterial endosymbiosis in yeast that functioned as an endosymbiotic organelle and was stable for more than 40 generations. (106) Formation of these cellular compartmentalizations can be achieved in synthetic systems, such as polymer-based aqueous two-phase systems and hydrogels. (107−110)
Synthetic compartmentalization has been achieved through different techniques─e.g., encapsulation in amphiphilic lipids, peptides, or polymers, protein capsids, and liquid–liquid phase separation─and is technically relatively facile. Applied methods include, for example, thin-film hydration, biphasic centrifugation, extrusion, and sonication. (111) Generating liposomes containing genetic material is as simple as adding a solution of plasmid to a dried thin film of phospholipid. The challenge with compartmentalization has been the relatively low efficiency of encapsulating molecules at consistent quantities within compartments. Stochasticity often generates wide variation in the composition of individual liposomes encapsulating cell extract. (112) The size of the artificial compartments themselves can vary considerably depending on the method used, as can the relative spatial connectivity. These technical hurdles stymie the production of cell-like compartments with sufficient reproducibility to study their behavior. While microfluidic tools and methods are available, improved methods are needed to create compartments of consistent size, composition, and connectivity to enable more systematic and quantitative studies.
In many cases, it will be desirable to build compartments within other compartments and then manipulate those, for example, through fusion or fission. Forming multiple compartments within a larger liposome is possible by microfluidic double emulsion liposome construction using different reagents for the dewetting process. (113) Some researchers have encased live cells within liposomal cells to allow for compartments with different internal properties. (114) Genetic cascade reactions can be compartmentalized for controlled modular reactions. Incompatible reactions can be allocated to separate liposomes and then fused together to allow for concurrent reactions. (35)
A variety of other techniques, beyond the use of lipid membranes, have been demonstrated for compartmentalization. The most common alternative to membranes are water–oil emulsions created with microfluidics. Those systems enable the compartmentalization functionalities of liposomes, using membrane proteins. As these emulsions tend to be unstable outside of carefully controlled environments, their utility for applications, such as drug delivery, is currently limited. (115) Peptide-based compartments have also been used to contain biochemical reactions and reaction networks. (116−118) Single-stranded DNA has also been used to form complex flexible coacervates, and double-stranded DNA can favor coacervate formation in the presence of cationic polymers. (119,120) Additionally, low-complexity RNA molecules can reversibly compartmentalize peptides and oligonucleotides by modifying the temperature. (121) Zeolites, microporous crystalline aluminosilicates, can compartmentalize molecules of choice. (122) Understanding how membrane-less compartments are created within cells through self-organization will also suggest engineering strategies to build a cell. (123)

Metabolism

Metabolism is the chemical means by which life harnesses energy to organize and maintain itself in a nonequilibrium state at the cost of increasing the entropy of the external environment. This set of chemical reactions sustains life through the assimilation of substrate and harvesting of energy for conversion into compounds that allow the biosynthesis of cellular components.
Individual biochemical reactions can be straightforward to replicate. However, recreating a carefully balanced, self-sustaining network of biochemistry that mobilizes energy and matter and commits it to growth and replication is arguably one of the most difficult aspects of building a living cell. First, the construction and dynamic balancing of a complex system of catabolic and anabolic chemistries is by itself a formidable hurdle. For replication, a net-excess of matter (i.e., fixed carbon, nitrogen, and other elements) must be produced. Second, the metabolic system must be encoded genetically to propagate continuously, which creates the additional challenge of engineering the timing and amount of each component to be formed throughout the life-cycle of an organism. In known extant cells, metabolism must be coupled in some way to replication. Hence, metabolism depends on itself in what could be conceived as an arbitrarily complex differential equation. Despite this, primordial cells could have evolved with only a tenuous link between metabolism and replication. Metabolism may have resulted in passive cell division, mediated by forces exerted by membrane curvature.
To date, minimal synthetic metabolism has been demonstrated in various forms. Basic redox reactions and energy metabolism are key for cells to generate new chemical products, as well as their own energy for sustaining life. In most whole cells, adenosine triphosphate (ATP), the main energy-carrier of biology as we know it, is primarily regenerated by a trans-membrane ion-gradient that drives an ATP synthase. The presence of a membrane is therefore crucial in the function of these enzymes. However, production of synthetic membranes and correspondingly membrane-bound proteins, remains a challenge. Promisingly, cell-free expression can quickly generate membrane-bound proteins. (124) Alternatively, energy in the form of photons can be used to phosphorylate ADP. One method for achieving this is through the encapsulation of rhodopsin, a light-gated ion-pump, and ATP synthase. Encased in vesicles together, they convert light energy into a proton-gradient that generates ATP via phosphorylation. (125) Other, cell-free systems have been developed that use pyruvate, (126) maltose, (127) 3-phosphoglyceric acid, (128) and phosphoenolpyruvate, (129) to provide energy to maintain biochemical reactions. This area has been extensively developed and is reviewed elsewhere. (130)

Replication

Replication requires the existence of genetic material that stores information and can be inherited by any progeny. This information minimally comprises instructions for replication, such as the genetic code of a virus. Achieving replication of a synthetic cell requires engineering of both a system for replicating the genetic material and a mechanism for subdividing a complete copy of the genetic material into a new compartment. Also needed is sufficient (bio)chemical activity to generate or scavenge resources for the synthesis of both the genetic material and the components of its containing compartment. Integration and coordination of each facet of replication is ultimately required to ensure its maintenance over generations.
Replication of internal biochemistry and cellular membranes is essential for cell perpetuation. Methods of accomplishing this include extrusion through primordial clay and cell-free expression of a minimal divisome. (131,132) Liposomes can be fused and fissured through a freeze–thaw method to generate larger liposomes, which can then be used for encapsulation of larger compounds. (133)
Nucleic acid polymers are currently the only known genetic material. DNA, the predominant form of genetic material on Earth, is highly stable (half-life of millennia) and dense in information. (134) RNA, which is prone to autocatalytic degradation and alkaline hydrolysis, has a shorter half-life in vivo (only minutes) than DNA but becomes more stable when bound to ribosomes. (135,136) Self-replicating RNAs have been demonstrated in multiple experiments. (137,138) DNA, however, requires extensive protein machinery to replicate, and deoxyribonucleotides are converted to ribonucleotides by ribonucleotide reductase. This has been a powerful argument for the existence of an “RNA world” prior to the evolution of DNA as the most widely used genetic material on Earth.
RNAs have been shown to retain catalytic activity when encapsulated in lipid vesicles. (17,139) Because of this, and because no autonomous self-replicating DNA is known to exist, RNA may be the more promising genetic material for a truly bottom-up synthetic cell in the near future. However, DNA-based replication should remain an engineering goal, because of its much greater stability. In addition, proofreading and repair is more common for double stranded DNA than for RNA: in vivo replication of DNA replication has an error rate of less than one per 100 million base pairs. Viral RNA replication and transcription of DNA produces an error every thousand bases. Furthermore, DNA molecules can be up to 250 million base pairs large, for example, in the human genome, whereas most RNAs are not more than a few thousand base pairs, with the current record length belonging to a nidovirus, which has an RNA genome of 41 thousand bases (single strand). (140) A DNA-based synthetic cell would require a number of additional factors, greatly increasing its complexity. For example, E. coli replication requires at minimum helicase, primase, DNA polymerase, and ligase, all of which would need to be encoded and expressed. This, in turn, requires machinery and raw material for transcription and translation to produce each enzyme. In addition, the progeny material must be produced faster than the parent material is degraded. (141) Still, a synthetic cell with a DNA genome that recapitulates features of Earth’s first cells might be much less complicated. Were helicase, primase, DNA polymerase, and ligase enzymes present in the first cell with a DNA genome, or was a simpler mechanism present in the progenitor?
Living cells have elaborate pathways that orchestrate the physical partitioning of copied genomes into new compartments. While careful partitioning of the old and copied genomes has evolved in most organisms, this is not strictly required. Multiple copies of genomes can persist in a single compartment and then be randomly segregated during division. This is the case for the majority of bacterial multicopy plasmids. (142) Without partitioning mechanisms, this would result in a high fraction of progeny having either none or multiple copies of genomes. As long as some fidelity (and diversity) is maintained across generations of synthetic cells, the population can persist. The spontaneous division of lipid vesicles has been demonstrated in multiple systems, however, none have been coupled to the replication of genetic material.
Box 1

Assembly of Artificial Genomes Based on Natural or Synthetic DNA

Assuming the first synthetic cell will rely on DNA as the genetic material, then this coding sequence must be either chemically synthesized or derived from an extant organism. The middle-out approach of using existing DNA from a living organism is appealing, because it does not require detailed design knowledge of the genetic code itself. De novo design of every base pair in even a simple genome is a daunting prospect, requiring holistic understanding of not only the function but also interaction of all genetic elements and gene regulatory networks. (143−147)
Hence, the coding of the first synthetic cell genome is likely to be naturally derived, while the physical DNA itself will almost certainly be chemically synthesized. This is due to the rapid progress that has been made in recent decades both in the reduction of the cost of synthetic DNA and technical leaps that have enabled stitching relatively small oligonucleotides into massive assemblies of genes, chromosomes, and even whole genomes. The size of the DNA for the first synthetic cell could vary wildly from a few thousand to a few million base pairs. A quarter of a century ago, the first synthetic genome, an 8 kbp DNA copy of a hepatitis C virus subgenomic replicon, took months to assemble from synthetic oligonucleotides. (148) Less than a decade later, scientists had developed DNA synthesis technologies that allowed completion of the 583 kbp Mycoplasma genitalium genome. (30) This became possible with the invention of homology-based assembly strategies relying on chemically synthesized subgenomic DNA fragments of 1 to 5 kbp. (149) The fragments we recombined into shuttle-vectors and then transformed into Escherichia coli to obtain assemblies of 10 to 30 kbp. In a second assembly step, these fragments were transformed into yeast where homologous recombination allowed formation of artificial chromosomes of 20 to 140 kbp. Outgoing from these, fragments can be isolated and used to create a living or nonliving synthetic cell. (150)
The booting-up process of synthetic genomes is not trivial, and genome transplantation has only been accomplished for the species Mycoplasma. (29,31,32) The only other organism for which a completely synthetic genome has been loaded is E. coli. (65,151) Additionally, Saccharomyces cerevisiae strains with multiple synthetic chromosomes have been made where native genomes are replaced with corresponding ∼50 kbp synthetic segments that are iteratively transformed into cells where the swap takes place. (65,151)
While the cost for the construction of large DNA fragments have decreased substantially with the emergence of DNA foundries, transplantation of nonmycoplasma genomes via a similar or equivalent universally applicable technique is still limited to nonliving cells with coding sequences that are much less complex than those of natural cells. (150,152)

Emergent Features of Life

Evolution

Evolution is a process that emerges when a population of individuals shows differential survival based on a heritable phenotype; an individual cell does not evolve. For synthetic cell engineers, this means that evolution cannot be observed to occur unless a population of synthetic cells with a heritable phenotype has been created. Evolution also requires that the organism’s phenotype is the result of its genotype. The evolution of individuality, referred to as the “Darwinian threshold”, (153,154) was key to the origin of predominantly vertical rather than horizontal transmission of genetic material.
For example, in the case of the JCVI minimal bacterial cell, it was hypothesized that a bacterium whose genome only encoded genes essential for cell-viability might be unable to evolve, mainly because of the absence of DNA-repair mechanisms that would lead to the rapid accumulation of fatal mutations. This idea was disproved recently in adaptive laboratory evolution experiments, demonstrating increased fitness of JCVIsyn3A and JCVI-syn3B after thousands of generations in laboratory culture. (155,156)
Evolution should inevitably emerge from a population of individual cells where there is some relation of phenotype to heredity, whether by natural selection, drift, or another mechanism. If the environment changes or more than one organism competes for a resource, taxa must evolve to even “stand still”, according to van Valen’s “Red Queen Hypothesis”. (157,158) Evolution requires individuals, so it can occur at subcellular levels as well, where mitochondria and chloroplasts can count as individuals, all the way down to the genes. For example, the usage of NSAAs may arise that result in new and diverse chemical properties of proteins. This may expand the functional ability of proteins, resulting in a potential evolutionary advantage to the host cell. (159)

Responsiveness

Sensing stimuli and responding by altering system behavior is an emergent trait of life. This enables organisms to adjust their behavior to a changing environment. The unique ability of cells to sense and respond to stimuli has provided inspiration for engineers, for example, in the creation of biomimetic materials.
Communication among populations is vital for population-level adaptation from unicellular organisms to entire mixed ecologies. Understanding intercell communication systems has enabled applications in synthetic biology. (160) Mechanisms for cellular sensing and responding are essential for the coordination of spatially separated functional modules. Advancements in programmable cell communication are expected to enable advanced control over synthetic cells. For example, cells with a porous membrane may utilize nucleus-like DNA hydrogel to express signaling molecules to communicate with neighboring cells. Utilizing this technology enables the creation of mutualisms. (161) Programmable mechanosensitivity in synthetic cells has been accomplished using osmotic pressure through mechanosensitive channels. (162) Further, advancements have been made in the areas of signaling molecules, signal range, active and passive signaling, (163) and information transfer, (164) as discussed previously. (165)
It can also be argued that responsiveness to environmental change is not a necessary property of living cells, at least in captivity. In nature, the bacteria with the smallest genomes tend to be obligate parasites. Bacteria like mycoplasmas, which are human urogenital pathogens, are examples of this. (166) They evolved from bacteria like Bacillus subtilis through a process of massive gene loss. (167) This was likely possible due to a very stable and nutritionally-rich habitat. Human urogenital epithelial cells, which these bacteria parasitize, provide such an environment. One can imagine that over years of completely constant laboratory culture, these mycoplasma species would be able to also discard all the approximately 60 genes they have retained for growth in their natural environment to deal with changes in urine osmolarity or the presence of other parasitic bacteria. A synthetic cell incapable of responding to its physical, chemical, or biological environment could be highly desirable: if it were to be released into the environment, it would presumably not be able to survive, therefore providing intrinsic biocontainment.

Movement

While directed movement, whether on a micro- or macroscale, is not required for life, many organisms demonstrate some form of mobility. (168) For most, movement is necessary to locate and obtain resources, to avoid living in a buildup of waste-products, to find a mate, or avoid predation. In microorganisms, flagella, microvilli, membrane blebbing, and gliding activity enable and facilitate movement. (169) Some organisms are small enough that ambient fluid flow or Brownian motion is sufficient to support their molecular processes. As such, even growth and division can be classified as movement─by that definition all living cells experience microscopic movement.
While directed movement can be thought of as a form of responsiveness, because the movement is often in response to some stimuli, the molecular machinery that enables movement can be distinct from the responsive elements. A greater understanding of the relationship between morphology and motility allows for increased ingenuity in creating artificial movement. The specific strategies for creating cellular protrusions, substrate adhesion, and myosin-dependent actin network contractility in synthetic cells are discussed elsewhere. (170)
Each of the above properties of living organisms have been engineered into synthetic systems to varying degrees, sometimes even in (albeit limited) combinations. In theory, once the criteria of compartmentalization, replication, and metabolism are established simultaneously, a new lifeform should emerge. Table 2 summarizes the state of efforts to date in that regard.
Table 2. Efforts Undertaken toward Building Synthetic Cells to Datea
titleDOIyearlabapproachgenetic replicationdivisioncompartmentalizationmetabolismmovementsense and respondevolution
Oparin’s Reactions Revisited: Enzymatic Synthesis of Poly(adenylic acid) in Micelles and Self-Reproducing Vesicles10.1021/ja00096a0101994LuisiBottom-upYesYesYesNoNoNoNo
Man-made cell-like compartments for molecule evolution10.1038/nbt0798-6521998GriffithMiddle-outNoNoYesNoNoNoNo
A vesicle bioreactor as a step toward an artificial cell assembly10.1073/pnas.0408236102004LibchaberBottom-upNoNoYesYesNoNoNo
Design of artificial cell–cell communication using gene and metabolic networks10.1073/pnas.03064841012004LiaoTop-downNoNoYesYesNoYesNo
Self-maintained Movements of Droplets with Convection Flow10.1007/978-3-540-76931-6_162007IkegamiBottom-upNoNoNoNoYesYesNo
Multilevel Selection in Models of Prebiotic Evolution II: A Direct Comparison of Compartmentalization and Spatial Self-Organization10.1371/journal.pcbi.10005422009HogewegTop-downYesNoYesNoNoNoYes
Creation of a bacterial cell controlled by a chemically synthesized genome10.1126/science.11907192010GibsonTop-downYesYesNoYesNoYesYes
Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA10.1038/nchem.11272011SugawaraBottom-upNoYesYesYes*NoNoNo
An Open Question on the Origin of Life: The First Forms of Metabolism10.1002/cbdv.2012002812012LuisiBottom-upNoNoYesYesNoNoNo
Spontaneous network formation among cooperative RNA replicators10.1038/nature115492012LehmanTop-downNoNoNoNoNoYesYes
Liposome division by a simple bacterial division machinery10.1073/pnas.12222541102013EricksonBottom-upNoYesYesNoNoNoNo
Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment10.1038/ncomms34942013YomoBottom-upYesNoYesNoNoNoYes
Protein synthesis in artificial cells: using compartmentalization for spatial organization in vesicle bioreactors10.1039/C4CP05933F2015CesBottom-upNoNoYesYesNoNoNo
Self Guided Supramolecular Cargo Loaded Nanomotors with Chemotactic Behavior toward Cells10.1002/anie.2015041862015van Hest and WilsonBottom-upNoNoYesNoYesNoNo
Design and synthesis of a minimal bacterial genome10.1126/science.aad62532016HutchisonTop-downYesYesNoYesYesYesYes
Growth and division of active droplets provides a model for protocells10.1038/nphys39842016JülicherBottom-upYesYesYesNoNoNoYes
Engineering genetic circuit interactions within and between synthetic minimal cells10.1038/nchem.26442017AdamalaMiddle-outNoNoYesNoNoYesNo
The origin of heredity in protocells10.1098/rstb.2016.04192017LaneBottom-upNoYesYesNoNoNoYes
Light-Guided Motility of a Minimal Synthetic Cell10.1021/acs.nanolett.8b034692018WegnerBottom-upNoNoYesNoYesYes*No
Enzyme-powered motility in buoyant organoclay/DNA protocells10.1038/s41557-018-0119-32018MannMiddle-outNoNoYesNoYesYesNo
Sustainable replication and coevolution of cooperative RNAs in an artificial cell-like system10.1038/s41559-018-0650-z2018Mizuuchi and IchihashiTop-downYesYesYesNoNoNoYes
Freeze–thaw cycles induce content exchange between cell-sized lipid vesicles10.1088/1367-2630/aabb962018SchwilleBottom-upNoNoYesNoNoNoNo
Self-replication of DNA by its encoded proteins in liposome-based synthetic cells10.1073/pnas.19146561172018DanelonBottom-upYesNoYesNoNoNoNo
Artificial photosynthetic cell producing energy for protein synthesis10.1038/s41467-019-09147-42019Ueda, KurumaBottom-upNoNoYesYesNoYesNo
Bottom-up Creation of an Artificial Cell Covered with the Adhesive Bacterionanofiber Protein AtaA10.1021/jacs.9b093402019Matsuura, HoriBottom-upNoNoYesNoNoNoNo
An Adaptive Synthetic Cell Based on Mechanosensing, Biosensing, and Inducible Gene Circuits10.1021/acssynbio.9b002042019NoireauxMiddle-outNoNoYesNoNoYesNo
Motility of Enzyme-Powered Vesicles10.1021/acs.nanolett.9b018302019SenBottom-upNoNoYesNoYesNoNo
Mimicking Chemotactic Cell Migration with DNA Programmable Synthetic Vesicles10.1021/acs.nanolett.9b044282019ChoiMiddle-out?NoNoYesNoYesYesNo
Chemical Signal Communication between Two Protoorganelles in a Lipid-Based Artificial Cell10.1021/acs.analchem.9b011282019HanMiddle-out?NoNoYesNoNoYesNo
Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration10.1021/acssynbio.0c001102020Biner and HirstBottom-upNoNoYesYesNoNoNo
Self-division of giant vesicles driven by an internal enzymatic reaction10.1039/C9SC05195C2020Lagzi, RossiBottom-upNoYesYesNoNoYesNo
Self Propelled PLGA Micromotor with Chemotactic Response to Inflammation10.1002/adhm.2019017102020WilsonBottom-upNoNoYesNoYesNoNo
A Step toward Molecular Evolution of RNA: Ribose Binds to Prebiotic Fatty Acid Membranes, and Nucleosides Bind Better than Individual Bases Do10.1002/cbic.2020002602020KellerBottom-upNoNoYesNoNoNoNo
Hydrodynamic accumulation of small molecules and ions into cell-sized liposomes against a concentration gradient10.1038/s42004-020-0277-22020ToyotaBottom-upNoNoYesNoNoNoNo
Dissipative self-assembly, competition and inhibition in a self-reproducing protocell model10.1039/D0SC02768E2020FletcherBottom-upYesNoYesNoNoNoNo
Catalytic processing in ruthenium-based polyoxometalate coacervate protocells10.1038/s41467-019-13759-12020MannMiddle-out?NoNoYesNoNoYesNo
Membrane molecular crowding enhances MreB polymerization to shape synthetic cells from spheres to rods10.1073/pnas.19146561172020NoireauxBottom-upNoNoYesYesNoNoNo
Signaling and differentiation in emulsion-based multi-compartmentalized in vitro gene circuits10.1038/s41557-018-0174-92020SimmelBottom-upNoNoYesNoNoYesNo
Engineering motile aqueous phase-separated droplets via liposome stabilization10.1038/s41467-021-21832-x2021CesBottom-upNoNoYesNoYesNoNod
Light-Powered Reactivation of Flagella and Contraction of Microtubule Networks: Toward Building an Artificial Cell10.1021/acssynbio.1c000712021GholamiMiddle-outNoNoYesYesYesYesNo
Reconstitution of contractile actomyosin rings in vesicles10.1038/s41467-021-22422-72021SchwilleBottom-upNoYesYesNoNoNoNo
Programmable Aggregation of Artificial Cells with DNA Signals10.1021/acssynbio.0c005502021ChoiBottom-upNoNoYesNoNoYesNo
Phase Separation and Protein Partitioning in Compartmentalized Cell-Free Expression Reactions10.1021/acs.biomac.1c005462021MaedaMiddle-outNoNoYesNoNoNoNo
Light-Triggered Cargo Loading and Division of DNA-Containing Giant Unilamellar Lipid Vesicles10.1021/acs.nanolett.1c008222021GöpfrichBottom-upNoYesYesNoNoNoNo
Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multicompartment artificial cells10.1073/pnas.20121701182021MavelliMiddle-outNoNoYesYesNoYesNo
Programmable Fusion and Differentiation of Synthetic Minimal Cells10.1021/acssynbio.1c005192022AdamalaBottom-upNoNoYesYesNoNoYes
Signal-processing and adaptive prototissue formation in metabolic DNA protocells10.1038/s41467-022-31632-62022WaltherBottom-upNoNoYesNoNoYesNo
Living material assembly of bacteriogenic protocells10.1038/s41586-022-05223-w2022MannMiddle-outNoNoYesYesNoYesNo
In vitro assembly, positioning and contraction of a division ring in minimal cells10.1038/s41467-022-33679-x2022SchwilleBottom-upNoYesYesNoNoNoNo
Gene silencing and transfection in synthetic cells10.1002/bit.284222023AdamalaBottom-upNoNoYesYesNoNoYes
Synthesizing a minimal cell with artificial metabolic pathways10.1038/s42004-023-00856-y2023ImaiMiddle-outNoNoYesYesNoNoNo
Clonal Amplification-Enhanced Gene Expression in Synthetic Vesicles10.1021/acssynbio.2c006682023DanelonBottom-upYesNoYesYesNoNoNo
Engineering cellular communication between light-activated synthetic cells and bacteria10.1038/s41589-023-01374-72023BoothMiddle-outNoNoYesNoNoYesNo
Signal transduction across synthetic cell membranes, with nucleic acid and peptide signals10.1016/j.cels.2023.12.0082024AdamalaBottom-upNoNoYesYesNoYesYes
a

Classification of approach may be ambiguous in certain cases.

Technical Challenges

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Clearly, building synthetic cells faces significant technical challenges and is still a long way from achieving its declared objective. Box 2 provides a high-level overview of the remaining hurdles and outlines a sensible approach to overcome these. The remainder of this paper further defines the specific technical challenges, organized by the criteria of life as defined above.
Box 2

Steps toward Rational Bottom-up Design and Construction of Living Cells

To achieve the building of a cell, a series of milestones will have to be reached. While there are likely numerous routes to success, we anticipate the most rational approach to comprise the following six stages, to be accomplished in somewhat chronological order:
1.

Accomplish DNA replication in an encapsulated cell-free system

2.

Develop synthetic translation modules (synthetic ribosome; i.e., ribosomes making ribosomes, and the rest of the translation apparatus)

3.

Implement cycling/self-replicating Tx/Tl systems

4.

Achieve autonomous division of

a.

synthetic cellular compartment(s) without DNA

b.

machineries that segregate DNA

5.

Show adaptation/sense response response (adaptive homeostasis) of synthetic biochemical systems

6.

Revisit the definition of biological life to include synthetic biochemistry

Finally, an appropriate biosafety and -security framework will have to be instituted in parallel to and in correspondence with the progression of steps one to six that is applicable and pertinent to the new lifeform.
Since parallel development and cross-information on the six steps outlined above is highly likely, we have composed an overview of the interdependences in the path toward building of a cell in Figure 4.

Figure 4

Figure 4. Milestones toward the de novo design and construction of a synthetic cell. Many of these steps can be performed in parallel with a mixing and matching approach to integration.

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Compartmentalization

To achieve the formation of compartments, reliable methods for vesicular synthesis are required. Traditional means, such as thin-film hydration to encapsulate cell-free systems in liposomes, results in a large range in the size and shape of liposomes. (35,171) This leads to variable protein expression among liposomes. (112,172) Another source of variability, especially in small (<10 μm) liposomes, is the random encapsulation of reactants. (173) This randomness means that some liposomes contain sufficient reactants to produce protein as opposed to nominal content of others. Even with microfluidic encapsulation, which ensures more uniform size and content, macromolecular crowding can lead to large local variability within the same liposome. (174)

Metabolism

Functional metabolism requires the development of robust catabolic and anabolic processes for the mobilization of substrate and energy. This will allow for the biosynthesis of essential cellular compounds, such as carbohydrates, fatty acids, and amino acids, that are the building blocks to minimize the reliance on externally supplied nutrients.
Given the large amount of transcription and translation that must occur simultaneously in synthetic cells, temporal control over gene-expression is critical. Whole cells have evolved complex methods to regulate genes in order to allocate limited resources toward the necessary metabolic functions required to maintain life. This control is often maintained via a series of genetic circuits that interact with each other, such as feedback and -forward loops, as well as oscillators. Advancements have been made in developing orthogonal gene regulators, which allow for the control of multiple genes simultaneously. However, emulating the complex gene regulation of whole cells in a cell-free environment remains a challenge. Furthermore, as we progress into developing synthetic cells with extended capabilities, for example, to produce chemicals, allocation of resources becomes an even larger challenge, as there must be a balance between maintenance of cellular functions and formation of the product of interest. (175,176)

Transcription and Translation Systems

Cell-free transcription and translation systems have advanced capabilities of in vitro protein expression by creating controllable, tunable systems independent of cell viability. The ‘Protein synthesis Using Recombinant Elements’ (PURE) system comprises the minimum number of enzymes required for Tx/Tl under specific conditions of almost any given gene. The PURE components are expressed in vivo, purified individually, and then combined. (40) This artificially reconstituted cell-free translation system allows controlled and high-throughput protein synthesis in vitro. (177) As with many novel technologies, the cost of supplies is a significant barrier. Nevertheless, promising advances in generating low-cost methods to produce the PURE cell-free system have been made. (178) “PURE to make PURE” is an example of this. Further, while completely tunable, the system consumes many resources and therefore has a lower protein output than crude cell extracts. (179) Crude cell extracts are derived from lysed whole cells where insoluble constituents have been removed via centrifugation, yielding an extract that contains a concentrated mix of cytosolic proteins. (180,181) Further processing by methods, such as dialysis, can improve the performance of such systems. Maintaining transcription and translation over extended periods in either of these cell-free systems remains a challenge, as with no regeneration most transcription and translation processes terminate after a few hours due to depletion of resources. In order to overcome this, long-lasting transcription and translation systems have been developed that continually replenish resources, such as tRNAs, amino acids, and salts, via a feeding solution or exhibit partial endogenous metabolism (e.g., oxidative phosphorylation). (182,180)
Advanced cell-free protein synthesis systems can produce up to 0.5 mg/mL protein in 2 to 4 h. (183) Nevertheless, these systems pale in comparison to the capabilities of naturally evolved cells as intracellular protein concentrations can be orders of magnitude higher. (184) The protein concentration of the cytoplasm in E. coli, for example, can reach 320 mg/mL, depending on the osmotic concentration of the medium. (185,186) If assuming a doubling time of ∼20 min, a protein production rate of 960 mg/mL cytoplasm per hour is theoretically feasible.
The ribosome is the “molecular machine” at the core of the translation system. Understanding of the ribosome has been transformed by the determination of three-dimensional structures, single molecule studies, and the construction of ribosomes from in vitro synthesized parts. (187,188) Despite this understanding, bottom-up synthesis of the ribosome in vitro remains a challenge. To achieve this objective, which is critical to building a true synthetic cell from the bottom-up, several key-achievements are needed, including the synthesis and assembly of rRNA and rProteins. Recent strides have been made in the construction of semisynthetic ribosomes using iSAT (integrated rRNA (rRNA) synthesis, ribosome assembly, and translation technology) in ribosome-free, crude cell lysates. (189) iSAT ribosomes are capable of constructing ribosomes. (190) In contrast to previous methods, this approach mimics the in vivo environment, including the cotranscription of rRNA and ribosome assembly, followed by protein synthesis in the same compartment. Similar methods imitate a natural cell’s cytoplasmic environment to allow for the de novo synthesis of ribosomes. (191)
The translation system is composed of many parts (ribosomes, tRNAs, aaRS, etc.), and synthesizing all of these is an energy-intensive process. Biosynthesis of the E. coli ribosome alone requires 7,434 peptide bonds to make a complete set of rProteins. (189) A successfully self-replicating translation system would need to be able to not only replicate components of the translation system but also any essential auxiliary proteins. (192) Any such system would come close to achieving a living synthetic cell.

Replication

No cell is immortal, thus replication is key to the persistence of life, as well as to evolution and the creation of a sufficiently dense population that ensures survival. PCR has been used to replicate genetic material in liposomes since 1995. (193) Although the replication and division of genetic material in liposomes is still not spontaneous, this technology has built a strong foundation for coupling with other technologies that work toward the goal of a synthetic cell. In vivo studies of natural cells have helped to elucidate the mechanisms of cell division. The minimal division machinery relies on gene circuits, metabolism, and macromolecular modules that have evolved to function in the environment of a living cell, and thus are not easily transferable to synthetic cells. Nevertheless, major strides have been made to control and observe genome management and separation. Future reconstitution attempts using cellular components may need to create environments that mimic natural crowding and associated electrostatic and excluded volume effects, such as was observed for DNA acting as an exclusion zone for actin fibers encapsulated in beads. (194) Synthetic systems that enable segregation of genomes to complement cell division still need to be established. Minimal-systems, as well as the proto-ring components, must be encapsulated in vesicles or other deformable compartments. (195) Analysis of cell division in JCVI-syn3.0 showed that it does not divide like most normal cells. Because it lacks a set of seven nonessential genes that include the cell-division proteins FtsZ and SepF, the cell appears to divide based on forces of membrane curvature rather than by forming a protein–lipid membrane between daughter cells. A similar process may have existed before the evolution of more sophisticated cell-division mechanisms. (196) Thus, construction of synthetic cells with replication systems simpler than those in the vast majority of lifeforms on Earth may be possible.

Emergent Features

Evolution

Evolution relies on the differential selection of variants whose phenotype is hereditary. In other words, evolution should emerge as long as there is heredity coupled to a phenotype and some variability (heterogeneity) in the population. To unleash the potential of evolution in a synthetic cell, genetic material must be passed on through generations. This can be accomplished by designing a synthetic genome, which may utilize either RNA or DNA and could rely on isothermal replication thereof, analogous to existing cells.
The concept of an “RNA world”, which suggests that RNA performed as both the genetic information and replication machinery, serves as a common theory for the emergence of life on Earth. Efforts to develop this into a synthetic cellular system could validate the possibility of an RNA origin-of-life and provide a way to bottom-up construct a synthetic cell. However, a fully self-synthesizing ribozyme has yet to be discovered or engineered.

Responsiveness

Designing synthetic cells that can respond to specific stimuli without relying on simply altering gene-expression remains a significant challenge. There are a wide range of chemical reaction systems that are responsive to different stimuli. In biological systems, sometimes similarly responsive reactions take place that employ no genetic elements. Stimuli-responsive compartments could enable dynamic changes in the behavior and functionality of synthetic cells in response to external cues. Achieving this requires the development of responsive cellular components and mechanisms that can detect and transduce signals. (164)

Movement

Many current techniques for the creation of directional movement are focused on single enzyme mediated movement along a limited diversity of concentration gradients. More physics-based techniques using interfacial tension differences could also be incorporated into synthetic cell-like systems. Based on the Marangoni effect, liposome-stabilized cell-sized droplets have demonstrated negative chemotaxis, resulting in movement away from the stimulus. (197) Self-propelled Janus particles have also been tuned to deliver cargo in response to specific obstacle geometries. (198) However, linking these to complex information-processing pathways inside an artificial system has not yet been shown. While likely challenging, these would nonetheless be a promising route to providing a limited form of directional movement.
Alternatively, complex parts of living cells could be isolated and reconstituted in artificial cell-based systems, for example, cilia and flagella, following a middle-out approach. Methods will need to be developed for their incorporation in a synthetic cell and their linkage to the information processing system for providing more than mere random motion. Knowledge gained by the development of these techniques can result in a better understanding of the motion-creating machinery that is essential for cell movement, potentially providing information for their bottom-up reconstitution.
A third technique driving directional movement could be the encapsulation of a set of cytoskeletal elements with associated proteins, for example, by reconstituting actin network assembly. (199,200) Described as “gliding”, some species of mycoplasmas exhibit this feature, which provides shape transformations, but directional movement is limited.

Concurrent Challenges

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Integration with Natural Cells

Successful interfacing of artificial and natural cells requires the presence of robust communication pathways. In recent years, synthetic cell-driven quorum-sensing was used not only to achieve signaling responses in bacteria, but also to expand their sensory range via artificial cells acting as chemical translators. (201,202) In another approach, artificial cells encapsulating gene-networks were able to detect, interact, and kill bacteria in chemically diverse extracellular-like environments. (203) Lastly, interfacing with eukaryotic cells has been successfully demonstrated by cuboplex-mediated gene-silencing in Chinese Hamster Ovary (CHO) cells, promoting the differentiation of neural stem cells from an artificially synthesized and released neurotrophic factor. (204)

Integration and Scalability

Overall, challenges of scalability of synthetic cells exist at three levels: scalability of individual components (e.g., scaling the number of genes that can be expressed in a cell-free system), scalability of number of components that can be integrated (e.g., adding a mobility module to membrane protein modules), and the scaling of the replication cycles and culture volume. A standardized workflow can help to establish both vertically- and horizontally integrated technology stacks for robust, reliable construction of synthetic cells. Such a workflow may also be helpful to define the components of a synthetic cell at varying levels of complexity and create modular abstraction for prediction and design. One such abstraction, of containers and content, has been proposed and others may emerge. (205−207) Scalability of synthetic cells for applications will depend on the success of these efforts at abstraction, coordination, and integration.

Cross-Lab and Larger-Scale Coordination

To be successful in building a cell, the free and open-access flow of data, protocols, and probably also people will be required. Effective communication and coordination between individual researchers and groups is crucial for establishing and maintaining functioning collaborations. This is especially important as cells require multiple interacting components, which exceeds the capacity of individual laboratories. Several such groups exist, including the NSF-funded Research Coordination Network (RCN) “Build-a-Cell”. (208) Build-a-Cell is an open, international collaboration supporting the science and engineering of synthetic cells. Members of the Build-a-Cell research community bring expertise from a variety of fields and backgrounds to foster open channels of collaboration, coordinate fundraising efforts, address biosafety and biosecurity concerns, encourage open technology transfer, and organize outreach efforts to increase understanding among researchers, policymakers, and the public. Other groups, such as the German-led MaxSynBio, (209) the Dutch-led BasyC, (210) and efforts from related fields, exist. (211−213)
To realize the full potential of synthetic cells for biomanufacturing, advanced tools and infrastructure for scale-up will be required. U.S. legislation, such as the “Inflation Reduction Act of 2022” (214) and the White House Executive Order 14081 on “Advancing Biotechnology and Biomanufacturing Innovation for a Sustainable, Safe, and Secure American Bioeconomy”, (215) as well as U.S. government efforts, such as BioMADE, (216) could support infrastructure for manufacturing synthetic cells.

Modular Roadmapping for Individual Components

Developing a modular roadmap for the integration of existing technologies, such as DNA synthesis, nanopore technology, microelectromechanical systems, microfluidics, compact hyperspectral imaging and ML, generative AI, and quantum computing, will help to motivate the community toward greater action, focus resources, and accelerate progress. This roadmap must be versatile enough to incorporate new technologies as they emerge. Academic efforts to produce devices for constructing cells are as of yet mostly uncoordinated.

Biosafety and Regulatory Guidelines

The field of synthetic cell research faces not only technical but also social, ethical, and philosophical challenges to define what is life, along with key obstacles requiring prudence for advancing both synthetic cell research specifically and biotechnology as a whole. Future applications, social and political frameworks, and governance for synthetic cell technologies demand thoughtful consideration, to ensure benefits while avoiding risks and preventing hazards. Equitable governance and regulatory participation are crucial for biosafety and threat mitigation as synthetic cell technologies become more accessible, reducing the risk of (intentionally or unintentionally) harmful biology. Existing methodologies for risk assessment, especially related to synthetic biology, must be revised or established anew, considering potential hazards through red teaming events and promoting biocontainment strategies.
Ensuring safe market-integration of synthetic cell technologies necessitates efficient regulatory pipelines, boosting private investments and career interests. Standardized definitions are prerequisites for policy protocols, aiding clear communication of standard operating procedures (SOPs) and safety protocols. FDA approval, a lengthy process for drugs, lacks suitable components for assessing synthetic cell safety due to their developmental stage. Classification of synthetic cell laboratories under BioSafety Levels (BSL) provides a standardized foundation for safety measures. BSL ratings, ranging from 1 to 4, determine containment needs based on pathogen type, a model that is likely also suitable for synthetic cells. Incorporating synthetic cells into existing pathogen definitions would simplify the process, assessing them similarly to naturally evolved pathogens based on infection capabilities or reproductive rates.

Technology Transfer

To optimize the development of impactful synthetic biology tools for synthetic cells, it is crucial to emphasize knowledge and technology transfer, given the significant expert hours required. Sharing procedures and protocols fosters effective collaboration, reducing redundancy and unnecessary competition among researchers. A coordinated effort to integrate the technology necessary for modeling, making, and measuring synthetic cells will not only enhance collaboration but also facilitate continuous iteration and improvement of underlying technologies. This collaborative approach should include complete reporting of attempted approaches, especially those that do not yield satisfactory results, to prevent researchers from inadvertently duplicating previous failures. Establishing a framework for sharing these unpublished results could significantly increase research efficiency.
Research could, for example, follow the lead of software development and create an open-source model for modular “à la carte” biological parts to develop a comprehensive synthetic cell chassis. This not only allows for a shared and collaborative understanding of basic biology functions but also avoids limiting the definition of “life” and thus limiting the scope of the field.

Outreach and Education

Synthetic cells offer significant potential for biotechnology, but engaging the public in research and technology development is crucial. Emphasizing education with both policymakers and the public will not only support effective policy frameworks but also enhance the acceptance and impact of new technologies. Fostering a better grasp of and involvement in synthetic cell technologies will aid in cultivating a market. Enhancing science literacy in the realm of synthetic biology will ensure a qualified workforce for this rapidly advancing field.
Comprehensive outreach and knowledge dissemination are essential across all levels to develop research tools, establish clear communication with policymakers, and create a market and workforce conducive to applying new technologies. Notably, BioBits kits have already captured student interest, fostering greater understanding. (217−221) Elevating science literacy through do-it-yourself (DIY) communities not only facilitates ethical discussions but also enhances public awareness and comprehension of ongoing research and eventual technologies.
Leveraging community-based laboratories alongside newly established foundations for global synthetic biology education can effectively drive public engagement and education in synthetic cell technology. (222) These endeavors to broaden STEM education can be interconnected with synthetic cell research.

Accessibility and Equity

Both living and nonliving synthetic cells will become a larger and larger part of biotechnology worldwide. The economic impact of this technology could be huge. A sustainable bioeconomy has the potential to enable communities around the world to be self-sufficient, self-determined, and resilient. As a highly impactful biotech field still in its nascency, synthetic cell research is well positioned to address historical barriers to access and address equity and inclusion in science more deliberately and democratically. This includes efforts in expanding the reach of education, developing technologies for use across geographic and socioeconomic boundaries, and engaging across disciplines to make decisions about the future of synthetic cell technology. (210)

Conclusions and Steps Ahead

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Due to the opportunity to tailor cellular systems for a specific purpose, synthetic cells could have a wide range of potential applications in various fields, such as cell biology, astrobiology and origin-of-life, bioengineering and biomedicine, biomanufacturing, and bioprocess engineering. However, achieving a self-sustaining biochemical reaction remains a significant challenge: the development of a fully functional synthetic cell is a complex and ongoing scientific endeavor that requires the integration of multiple disciplines and technologies.
Accomplishing the de novo construction of cells will be an enormous achievement, especially if the devised system is capable of reproduction, which will be a key first─but not ultimate─achievement. Any first iteration of a synthetic cell will likely be very basic, lacking substantial versatility in its metabolism and environmental robustness, and hence difficult to keep alive. In summary, the development of synthetic cells is a complex and multifaceted endeavor that will require interdisciplinary collaborations and significant investment in both resources and infrastructure.

When and How to Declare Success

Finally, at what point do we declare success? How do we know if we have created a synthetic cell that is alive rather than a vesicle that just exhibits life-like functions? Following Gánti’s Chemoton model of life, at a minimum, the characteristics of compartmentalization, metabolism, and replication must be present.
Is translation, transcription, or cell division required? Mature mammalian red blood cells, for example, do not have a nucleus or ribosomes and thus are not capable of transcription, translation or cell division. How about derived characteristics such as evolution? Clearly, these cells evolved from other kinds of cells, and the evolution of the nucleated cells that give rise to red blood cells will change the red blood cells. The list of exceptions goes on. If we turn to naturally evolved cells, we can easily find counterexamples for each of the current criteria of life, because life is a population-level phenomenon. As it is impossible to account for all possible variations and no “natural definition” of life exists, it may be a matter of creating a cell that is “generally recognized as alive” or GRAA. This allows the field to progress independently of philosophical agreement on a definition of life. Thus, the definition of success is likely context dependent and will have to be adapted as science progresses.

Author Information

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  • Corresponding Author
  • Authors
    • Nils J. H. Averesch - Department of Civil and Environmental Engineering, Stanford University, Stanford, California 94305, United States;  Orcidhttps://orcid.org/0000-0002-6389-2646
    • Elizabeth A. Strychalski - National Institute of Standards and Technology, Gaithersburg, Maryland 20899, United States
    • Felix Moser - Synlife, One Kendall Square, Cambridge, Massachusetts 02139-1661, United States
    • John I. Glass - J. Craig Venter Institute, La Jolla, California 92037, United States;  Orcidhttps://orcid.org/0000-0002-3784-5301
    • Rolando Cruz Perez - Department of Bioengineering, Stanford University, Stanford, California 94305, United States;  Blue Marble Space Institute of Science at NASA Ames Research Center, Moffett Field, California 94035-1000, United States
    • Ibrahim O. Yekinni - Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, United States;  Orcidhttps://orcid.org/0000-0003-0727-180X
    • Brooke Rothschild-Mancinelli - School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0150, United States
    • Garrett A. Roberts Kingman - NASA Postdoctoral Program, Ames Research Center, Moffett Field, California 94035-1000, United States
    • Feilun Wu - J. Craig Venter Institute, Rockville, Maryland 20850, United States
    • Jorik Waeterschoot - Mechatronics, Biostatistics and Sensors (MeBioS), KU Leuven, 3000 Leuven Belgium;  Orcidhttps://orcid.org/0000-0002-3875-329X
    • Ion A. Ioannou - Department of Chemistry, MSRH, Imperial College London, London W12 0BZ, U.K.;  Orcidhttps://orcid.org/0000-0001-8749-1049
    • Michael C. Jewett - Department of Bioengineering, Stanford University, Stanford, California 94305, United States;  Orcidhttps://orcid.org/0000-0003-2948-6211
    • Allen P. Liu - Mechanical Engineering & Biomedical Engineering, Cellular and Molecular Biology, Biophysics, Applied Physics, University of Michigan, Ann Arbor, Michigan 48109, United States;  Orcidhttps://orcid.org/0000-0002-0309-7018
    • Vincent Noireaux - Physics and Nanotechnology, University of Minnesota, Minneapolis, Minnesota 55455, United States;  Orcidhttps://orcid.org/0000-0002-5213-273X
    • Carlise Sorenson - Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, United States
    • Katarzyna P. Adamala - Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, United States;  Orcidhttps://orcid.org/0000-0003-1066-7207
  • Author Contributions

    LJR, NJHA, FM, and KPA formulated the concept and structure of the article. LJR, NJHA, EAS, FM, JIG, RCP, IOY, BRM, GARK, FW, JW, IAI, MCJ, APL, VN, CS, and KPA wrote portions of the manuscript. LJR, EAS, FW, and JW created the figures. All authors edited the manuscript.

  • Notes
    Certain commercial entities, equipment, or materials may be identified in this document to describe an experimental procedure or concept adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the entities, materials, or equipment are necessarily the best available for the purpose.
    The authors declare no competing financial interest.

Acknowledgments

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We are grateful to every current or previous member of the “Build-a-Cell Roadmap Working Group” who drafted or edited this paper but chose not to be coauthors. NSF Research Coordination Network (RCN) funding (award number 1901145) supported this endeavor. In addition, LJR was supported by NASA’s Planetary Science Division Research Program, through the ISFM work package “Evolutionary Processes that Drove the Emergence and Early Distribution of Life” at NASA Ames Research Center. MCJ acknowledges funding from NSF Division of Chemical, Bioengineering, Environmental, and Transport Systems (award number 1936789.). APL acknowledges funding from NSF Division of Emerging Frontiers (award number 1935265).

Abbreviations

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BSL

BioSafety Levels

DBTL

Design-Build-Test-Learn

DIY

do-it-yourself

GRAA

generally recognized as alive

iSAT

integrated synthesis, assembly, and translation

NSAA

nonstandard amino acid

PCR

polymerase chain reaction

PURE

protein synthesis using recombinant elements

RCN

Research Coordination Network

SOP

standard operating procedure

STEM

science, technology, engineering and mathematics

Tx/Tl

transcription/translation

UBP

unnatural base pair

XNA

xenonucleic acid.

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    The article highlights how the process of engineering biol. systems has contributed to our understanding of how endogenous systems are put together and function - from a quant. description of gene expression and signal transduction to controlling spatial organization and cell-cell interactions. An important aim of synthetic biol. is to uncover the design principles of natural biol. systems through the rational design of gene and protein circuits. Here, we highlight how the process of engineering biol. systems - from synthetic promoters to the control of cell-cell interactions - has contributed to our understanding of how endogenous systems are put together and function. Synthetic biol. devices allow us to grasp intuitively the ranges of behavior generated by simple biol. circuits, such as linear cascades and interlocking feedback loops, as well as to exert control over natural processes, such as gene expression and population dynamics.
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  16. 16
    Rothschild, L. J. The Role of Emergence in Biology; Oxford University Press, 2008.
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    Adamala, K.; Szostak, J. W. Nonenzymatic Template-Directed RNA Synthesis inside Model Protocells. Science 2013, 342 (6162), 1098– 1100,  DOI: 10.1126/science.1241888
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    Nonenzymatic Template-Directed RNA Synthesis Inside Model Protocells
    Adamala, Katarzyna; Szostak, Jack W.
    Science (Washington, DC, United States) (2013), 342 (6162), 1098-1100CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Efforts to recreate a prebiotically plausible protocell, in which RNA replication occurs within a fatty acid vesicle, have been stalled by the destabilizing effect of Mg2+ on fatty acid membranes. Here we report that the presence of citrate protects fatty acid membranes from the disruptive effects of high Mg2+ ion concns. while allowing RNA copying to proceed, while also protecting single-stranded RNA from Mg2+-catalyzed degrdn. This combination of properties has allowed us to demonstrate the chem. copying of RNA templates inside fatty acid vesicles, which in turn allows for an increase in copying efficiency by bathing the vesicles in a continuously refreshed soln. of activated nucleotides.
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  18. 18
    Deamer, D.; Dworkin, J. P.; Sandford, S. A.; Bernstein, M. P.; Allamandola, L. J. The First Cell Membranes. Astrobiology 2002, 2 (4), 371– 381,  DOI: 10.1089/153110702762470482
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    The first cell membranes
    Deamer, David; Dworkin, Jason P.; Sandford, Scott A.; Bernstein, Max P.; Allamandola, Louis J.
    Astrobiology (2002), 2 (4), 371-381CODEN: ASTRC4; ISSN:1531-1074. (Mary Ann Liebert, Inc.)
    A review. Org. compds. are synthesized in the interstellar medium and can be delivered to planetary surfaces such as the early Earth, where they mix with endogenous species. Some of these compds. are amphiphilic, having polar and nonpolar groups on the same mol. Amphiphilic compds. spontaneously self-assemble into more complex structures such as bimol. layers, which in turn form closed membranous vesicles. The 1st forms of cellular life required self-assembled membranes that were likely to have been produced from amphiphilic compds. on the prebiotic Earth. Lab. simulations show that such vesicles readily encapsulate functional macromols., including nucleic acids and polymerases. The goal of future investigations will be to fabricate artificial cells as models of the origin of life.
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    Deamer, D. The Role of Lipid Membranes in Life’s Origin. Life 2017, 7 (1), 5,  DOI: 10.3390/life7010005
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    Discher, B. M.; Bermudez, H.; Hammer, D. A.; Discher, D. E.; Won, Y.-Y.; Bates, F. S. Cross-Linked Polymersome Membranes: Vesicles with Broadly Adjustable Properties. J. Phys. Chem. B 2002, 106 (11), 2848– 2854,  DOI: 10.1021/jp011958z
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    Cross-linked Polymersome Membranes: Vesicles with Broadly Adjustable Properties
    Discher, Bohdana M.; Bermudez, Harry; Hammer, Daniel A.; Discher, Dennis E.; Won, You-Yeon; Bates, Frank S.
    Journal of Physical Chemistry B (2002), 106 (11), 2848-2854CODEN: JPCBFK; ISSN:1089-5647. (American Chemical Society)
    Massively cross-linked and property-tunable membranes have been fabricated by free radical polymn. of self-assembled, block copolymer vesicles - polymersomes. Similar efforts with cross-linkable lipids would appear frustrated in the past due to at least two factors: limited reactivity and membrane fragility under local stresses of nano-confined crosslinking. We describe here a diblock copolymer of poly(ethylene oxide)-polybutadiene that has a hydrophilic wt. fraction like that of lipids and forms robust fluid phase membranes in water. The polymersomes sustain free radical polymn. of the hydrophobic butadiene, thereby generating a semipermeable nano-shell. Cross-linked giant vesicles prove stable in chloroform and can also be dehydrated and re-hydrated without rendering the ∼9 nm thick membrane core; the results imply defect-free membranes many microns-squared in area. Surface elastic moduli as well as sustainable wall stresses up to 103 Atm, orders of magnitude greater than any natural lipid membrane, appear consistent with strong tethering between close-packed neighbors. The enormous stability of the giant vesicles can be tuned down for application: blending in the hydrogenated analog poly(ethylene oxide)-polyethylethylene modulates the effective elastic consts. as well as the rupture strength by orders of magnitude. The results appear consistent with rigidity percolation through a finite-layer stack of two-dimensional lattices. Moreover, below the percolation limit, a regime of hyper-instability emerges, reflecting perhaps nanoscale demixing and suggestive of the limitations encountered with low reactivity lipids. The results provide general insights into covalent crosslinking within self-assembled nanostructures.
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  21. 21
    Kamat, N. P.; Katz, J. S.; Hammer, D. A. Engineering Polymersome Protocells. J. Phys. Chem. Lett. 2011, 2 (13), 1612– 1623,  DOI: 10.1021/jz200640x
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    Engineering Polymersome Protocells
    Kamat, Neha P.; Katz, Joshua S.; Hammer, Daniel A.
    Journal of Physical Chemistry Letters (2011), 2 (13), 1612-1623CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    A review. The field of biomimicry is embracing the construction of complex assemblies that imitate both biol. structure and function. Advancements in the design of these mimetics have generated a growing vision for creating an artificial cell or protocell. Polymersomes are vesicles that can be made from synthetic, biol., or hybrid polymers and can be used as a model template to build cell-like structures. In this perspective, we discuss various areas where polymersomes have been used to mimic cell functions as well as areas in which the synthetic flexibility of polymersomes would make them ideal candidates for a biomembrane mimetic. Designing a polymersome that comprehensively displays the behaviors discussed herein has the potential to lead to the development of an autonomous, responsive particle that resembles the intelligence of a biol. cell.
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    Vickers, C. E. The Minimal Genome Comes of Age. Nat. Biotechnol. 2016, 34 (6), 623– 624,  DOI: 10.1038/nbt.3593
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    Xu, X.; Meier, F.; Blount, B. A.; Pretorius, I. S.; Ellis, T.; Paulsen, I. T.; Williams, T. C. Trimming the Genomic Fat: Minimising and Re-Functionalising Genomes Using Synthetic Biology. Nat. Commun. 2023, 14 (1), 1984,  DOI: 10.1038/s41467-023-37748-7
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    23
    Trimming the genomic fat: minimising and re-functionalising genomes using synthetic biology
    Xu, Xin; Meier, Felix; Blount, Benjamin A.; Pretorius, Isak S.; Ellis, Tom; Paulsen, Ian T.; Williams, Thomas C.
    Nature Communications (2023), 14 (1), 1984CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
    A review. Naturally evolved organisms typically have large genomes that enable their survival and growth under various conditions. However, the complexity of genomes often precludes our complete understanding of them, and limits the success of biotechnol. designs. In contrast, minimal genomes have reduced complexity and therefore improved engineerability, increased biosynthetic capacity through the removal of unnecessary genetic elements, and less recalcitrance to complete characterization. Here, we review the past and current genome minimisation and re-functionalisation efforts, with an emphasis on the latest advances facilitated by synthetic genomics, and provide a crit. appraisal of their potential for industrial applications.
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  24. 24
    Hutchison, C. A.; Peterson, S. N.; Gill, S. R.; Cline, R. T.; White, O.; Fraser, C. M.; Smith, H. O.; Venter, J. C. Global Transposon Mutagenesis and a Minimal Mycoplasma Genome. Science 1999, 286 (5447), 2165– 2169,  DOI: 10.1126/science.286.5447.2165
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    24
    Global transposon mutagenesis and a minimal mycoplasma genome
    Hutchison, Clyde A., III; Peterson, Scott N.; Gill, Steven R.; Cline, Robin T.; White, Owen; Fraser, Claire M.; Smith, Hamilton O.; Venter, J. Craig
    Science (Washington, D. C.) (1999), 286 (5447), 2165-2169CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Mycoplasma genitalium with 517 genes has the smallest gene complement of any independently replicating cell so far identified. Global transposon mutagenesis was used to identify nonessential genes in an effort to learn whether the naturally occurring gene complement is a true minimal genome under lab. growth conditions. The positions of 2209 transposon insertions in the completely sequenced genomes of M. genitalium and its close relative M. pneumoniae were detd. by sequencing across the junction of the transposon and the genomic DNA. These junctions defined 1354 distinct sites of insertion that were not lethal. The anal. suggests that 265 to 350 of the 480 protein-coding genes of M. genitalium are essential under lab. growth conditions, including about 100 genes of unknown function.
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  25. 25
    Glass, J. I.; Assad-Garcia, N.; Alperovich, N.; Yooseph, S.; Lewis, M. R.; Maruf, M.; Hutchison, C. A., 3rd; Smith, H. O.; Venter, J. C. Essential Genes of a Minimal Bacterium. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (2), 425– 430,  DOI: 10.1073/pnas.0510013103
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    25
    Essential genes of a minimal bacterium
    Glass, John I.; Assad-Garcia, Nacyra; Alperovich, Nina; Yooseph, Shibu; Lewis, Matthew R.; Maruf, Mahir; Hutchison, Clyde A., III; Smith, Hamilton O.; Venter, J. Craig
    Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (2), 425-430CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Mycoplasma genitalium has the smallest genome of any organism that can be grown in pure culture. It has a minimal metab. and little genomic redundancy. Consequently, its genome is expected to be a close approxn. to the minimal set of genes needed to sustain bacterial life. Using global transposon mutagenesis, gene disruption mutants were isolated and characterized for 100 different nonessential protein-coding genes. None of the 43 RNA-coding genes were disrupted. Herein, 382 of the 482 M. genitalium protein-coding genes were identified as essential, plus 5 sets of disrupted genes that encode proteins with potentially redundant essential functions, such as phosphate transport. Genes encoding proteins of unknown function constitute 28% of the essential protein-coding genes set. Disruption of some genes accelerated M. genitalium growth. The genome of M. genitalium G37 (ATCC 33530) was resequenced and found to differ from the previously sequenced version at 34 sites; the new sequences replaces the original M. genitalium genome sequence in GenBank/EMBL/DDBJ under accession no. L43967.
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  26. 26
    Hutchison, C. A., 3rd; Chuang, R.-Y.; Noskov, V. N.; Assad-Garcia, N.; Deerinck, T. J.; Ellisman, M. H.; Gill, J.; Kannan, K.; Karas, B. J.; Ma, L.; Pelletier, J. F.; Qi, Z.-Q.; Richter, R. A.; Strychalski, E. A.; Sun, L.; Suzuki, Y.; Tsvetanova, B.; Wise, K. S.; Smith, H. O.; Glass, J. I.; Merryman, C.; Gibson, D. G.; Venter, J. C. Design and Synthesis of a Minimal Bacterial Genome. Science 2016, 351 (6280), aad6253,  DOI: 10.1126/science.aad6253
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    Hashimoto, M.; Ichimura, T.; Mizoguchi, H.; Tanaka, K.; Fujimitsu, K.; Keyamura, K.; Ote, T.; Yamakawa, T.; Yamazaki, Y.; Mori, H.; Katayama, T.; Kato, J.-I. Cell Size and Nucleoid Organization of Engineered Escherichia Coli Cells with a Reduced Genome. Mol. Microbiol. 2005, 55 (1), 137– 149,  DOI: 10.1111/j.1365-2958.2004.04386.x
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    27
    Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome
    Hashimoto, Masayuki; Ichimura, Toshiharu; Mizoguchi, Hiroshi; Tanaka, Kimie; Fujimitsu, Kazuyuki; Keyamura, Kenji; Ote, Tomotake; Yamakawa, Takehiro; Yamazaki, Yukiko; Mori, Hideo; Katayama, Tsutomu; Kato, Jun-ichi
    Molecular Microbiology (2005), 55 (1), 137-149CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
    The minimization of a genome is necessary to identify exptl. the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large-scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphol. change. Based on the nucleoid localization obsd. with these mutants, we discuss the cellular nucleoid dynamics.
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  28. 28
    Pósfai, G.; Plunkett, G., 3rd; Fehér, T.; Frisch, D.; Keil, G. M.; Umenhoffer, K.; Kolisnychenko, V.; Stahl, B.; Sharma, S. S.; de Arruda, M.; Burland, V.; Harcum, S. W.; Blattner, F. R. Emergent Properties of Reduced-Genome Escherichia Coli. Science 2006, 312 (5776), 1044– 1046,  DOI: 10.1126/science.1126439
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    Lartigue, C.; Glass, J. I.; Alperovich, N.; Pieper, R.; Parmar, P. P.; Hutchison, C. A., 3rd; Smith, H. O.; Venter, J. C. Genome Transplantation in Bacteria: Changing One Species to Another. Science 2007, 317 (5838), 632– 638,  DOI: 10.1126/science.1144622
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    Genome transplantation in bacteria: changing one species to another
    Lartigue, Carole; Glass, John I.; Alperovich, Nina; Pieper, Rembert; Parmar, Prashanth P.; Hutchison, Clyde A., III; Smith, Hamilton O.; Venter, J. Craig
    Science (Washington, DC, United States) (2007), 317 (5838), 632-638CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    As a step toward propagation of synthetic genomes, the authors completely replaced the genome of a bacterial cell with one from another species by transplanting a whole genome as naked DNA. Intact genomic DNA from Mycoplasma mycoides large colony (LC), virtually free of protein, was transplanted into Mycoplasma capricolum cells by polyethylene glycol-mediated transformation. Cells selected for tetracycline resistance, carried by the M. mycoides LC chromosome, contain the complete donor genome and are free of detectable recipient genomic sequences. These cells that result from genome transplantation are phenotypically identical to the M. mycoides LC donor strain as judged by several criteria.
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  30. 30
    Gibson, D. G.; Benders, G. A.; Andrews-Pfannkoch, C.; Denisova, E. A.; Baden-Tillson, H.; Zaveri, J.; Stockwell, T. B.; Brownley, A.; Thomas, D. W.; Algire, M. A.; Merryman, C.; Young, L.; Noskov, V. N.; Glass, J. I.; Venter, J. C.; Hutchison, C. A., 3rd; Smith, H. O. Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma Genitalium Genome. Science 2008, 319 (5867), 1215– 1220,  DOI: 10.1126/science.1151721
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    30
    Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome
    Gibson, Daniel G.; Benders, Gwynedd A.; Andrews-Pfannkoch, Cynthia; Denisova, Evgeniya A.; Baden-Tillson, Holly; Zaveri, Jayshree; Stockwell, Timothy B.; Brownley, Anushka; Thomas, David W.; Algire, Mikkel A.; Merryman, Chuck; Young, Lei; Noskov, Vladimir N.; Glass, John I.; Venter, J. Craig; Hutchison, Clyde A., III; Smith, Hamilton O.
    Science (Washington, DC, United States) (2008), 319 (5867), 1215-1220CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chem. synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approx. 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-assocd. recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA mols. from chem. synthesized pieces and also from combinations of natural and synthetic DNA segments.
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  31. 31
    Lartigue, C.; Vashee, S.; Algire, M. A.; Chuang, R.-Y.; Benders, G. A.; Ma, L.; Noskov, V. N.; Denisova, E. A.; Gibson, D. G.; Assad-Garcia, N.; Alperovich, N.; Thomas, D. W.; Merryman, C.; Hutchison, C. A., 3rd; Smith, H. O.; Venter, J. C.; Glass, J. I. Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast. Science 2009, 325 (5948), 1693– 1696,  DOI: 10.1126/science.1173759
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    31
    Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast
    Lartigue, Carole; Vashee, Sanjay; Algire, Mikkel A.; Chuang, Ray-Yuan; Benders, Gwynedd A.; Ma, Li; Noskov, Vladimir N.; Denisova, Evgeniya A.; Gibson, Daniel G.; Assad-Garcia, Nacyra; Alperovich, Nina; Thomas, David W.; Merryman, Chuck; Hutchison, Clyde A., III; Smith, Hamilton O.; Venter, J. Craig; Glass, John I.
    Science (Washington, DC, United States) (2009), 325 (5948), 1693-1696CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    We recently reported the chem. synthesis, assembly, and cloning of a bacterial genome in yeast. To produce a synthetic cell, the genome must be transferred from yeast to a receptive cytoplasm. Here we describe methods to accomplish this. We cloned a Mycoplasma mycoides genome as a yeast centromeric plasmid and then transplanted it into Mycoplasma capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast genetic systems and then transplanted to produce a new strain of M. mycoides. These methods allow the construction of strains that could not be produced with genetic tools available for this bacterium. The complete, annotated sequence of the transplanted M. mycoides capri genome is deposited in GenBank/EMBL/DDBJ with accession no. CP001668.
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  32. 32
    Gibson, D. G.; Glass, J. I.; Lartigue, C.; Noskov, V. N.; Chuang, R.-Y.; Algire, M. A.; Benders, G. A.; Montague, M. G.; Ma, L.; Moodie, M. M.; Merryman, C.; Vashee, S.; Krishnakumar, R.; Assad-Garcia, N.; Andrews-Pfannkoch, C.; Denisova, E. A.; Young, L.; Qi, Z.-Q.; Segall-Shapiro, T. H.; Calvey, C. H.; Parmar, P. P.; Hutchison, C. A., 3rd; Smith, H. O.; Venter, J. C. Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science 2010, 329 (5987), 52– 56,  DOI: 10.1126/science.1190719
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    Creation of a bacterial cell controlled by a chemically synthesized genome
    Gibson, Daniel G.; Glass, John I.; Lartigue, Carole; Noskov, Vladimir N.; Chuang, Ray-Yuan; Algire, Mikkel A.; Benders, Gwynedd A.; Montague, Michael G.; Ma, Li; Moodie, Monzia M.; Merryman, Chuck; Vashee, Sanjay; Krishnakumar, Radha; Assad-Garcia, Nacyra; Andrews-Pfannkoch, Cynthia; Denisova, Evgeniya A.; Young, Lei; Qi, Zhi-Qing; Segall-Shapiro, Thomas H.; Calvey, Christopher H.; Parmar, Prashanth P.; Hutchison, Clyde A., III; Smith, Hamilton O.; Venter, J. Craig
    Science (Washington, DC, United States) (2010), 329 (5987), 52-56CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication. The complete, annotated synthetic genome sequence is deposited in GenBank/EMBL/DDBJ with accession no. CP002027.
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    Stano, P.; Luisi, P. L. Semi-Synthetic Minimal Cells: Origin and Recent Developments. Curr. Opin. Biotechnol. 2013, 24 (4), 633– 638,  DOI: 10.1016/j.copbio.2013.01.002
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    Semi-synthetic minimal cells: origin and recent developments
    Stano, Pasquale; Luisi, Pier Luigi
    Current Opinion in Biotechnology (2013), 24 (4), 633-638CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)
    A review. The notion of minimal cells refers to cellular structures that contain the minimal and sufficient complexity to still be defined as living, or at least capable to display the most important features of biol. cells. Here the authors briefly describe the lab. construction of minimal cells, a project within the broader field of synthetic biol. In particular the authors discuss the advancements in the prepn. of semi-synthetic cells based on the encapsulation of biochems. inside liposomes, illustrating from the one hand the origin of this research and the most recent developments; and from the other the difficulties and limits of the approach. The role of physicochem. understandings is greatly emphasized.
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  34. 34
    Olivi, L.; Berger, M.; Creyghton, R. N. P.; De Franceschi, N.; Dekker, C.; Mulder, B. M.; Claassens, N. J.; Ten Wolde, P. R.; van der Oost, J. Towards a Synthetic Cell Cycle. Nat. Commun. 2021, 12 (1), 4531,  DOI: 10.1038/s41467-021-24772-8
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    34
    Towards a synthetic cell cycle
    Olivi, Lorenzo; Berger, Mareike; Creyghton, Ramon N. P.; De Franceschi, Nicola; Dekker, Cees; Mulder, Bela M.; Claassens, Nico J.; ten Wolde, Pieter Rein; van der Oost, John
    Nature Communications (2021), 12 (1), 4531CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: Recent developments in synthetic biol. may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. Here, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this Perspective, we discuss potential paths towards an integrated synthetic cell cycle.
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    Adamala, K. P.; Martin-Alarcon, D. A.; Guthrie-Honea, K. R.; Boyden, E. S. Engineering Genetic Circuit Interactions within and between Synthetic Minimal Cells. Nat. Chem. 2017, 9 (5), 431– 439,  DOI: 10.1038/nchem.2644
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    Engineering genetic circuit interactions within and between synthetic minimal cells
    Adamala, Katarzyna P.; Martin-Alarcon, Daniel A.; Guthrie-Honea, Katriona R.; Boyden, Edward S.
    Nature Chemistry (2017), 9 (5), 431-439CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    Genetic circuits and reaction cascades are of great importance for synthetic biol., biochem. and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chem. reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-contg. synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biol. cascades, an essential step towards their ultimate programmability.
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  36. 36
    Fenz, S. F.; Sachse, R.; Schmidt, T.; Kubick, S. Cell-Free Synthesis of Membrane Proteins: Tailored Cell Models out of Microsomes. Biochim. Biophys. Acta 2014, 1838 (5), 1382– 1388,  DOI: 10.1016/j.bbamem.2013.12.009
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    Kroll, A. V.; Fang, R. H.; Zhang, L. Biointerfacing and Applications of Cell Membrane-Coated Nanoparticles. Bioconjugate Chem. 2017, 28 (1), 23– 32,  DOI: 10.1021/acs.bioconjchem.6b00569
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    Biointerfacing and Applications of Cell Membrane-Coated Nanoparticles
    Kroll, Ashley V.; Fang, Ronnie H.; Zhang, Liangfang
    Bioconjugate Chemistry (2017), 28 (1), 23-32CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    The cell membrane-coated nanoparticle is a biomimetic platform consisting of a nanoparticulate core coated with membrane derived from a cell, such as a red blood cell, platelet, or cancer cell. The cell membrane "disguise" allows the particles to be perceived by the body as the source cell by interacting with its surroundings using the translocated surface membrane components. The newly bestowed characteristics of the membrane-coated nanoparticle can be utilized for biol. interfacing in the body, providing natural solns. to many biomedical issues. This Review will cover the interactions of these cell membrane-coated nanoparticles and their applications within three biomedical areas of interest, including (i) drug delivery, (ii) detoxification, and (iii) immune modulation.
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  38. 38
    Gurramkonda, C.; Rao, A.; Borhani, S.; Pilli, M.; Deldari, S.; Ge, X.; Pezeshk, N.; Han, T.-C.; Tolosa, M.; Kostov, Y.; Tolosa, L.; Wood, D. W.; Vattem, K.; Frey, D. D.; Rao, G. Improving the Recombinant Human Erythropoietin Glycosylation Using Microsome Supplementation in CHO Cell-Free System. Biotechnol. Bioeng. 2018, 115 (5), 1253– 1264,  DOI: 10.1002/bit.26554
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    Su’etsugu, M.; Takada, H.; Katayama, T.; Tsujimoto, H. Exponential Propagation of Large Circular DNA by Reconstitution of a Chromosome-Replication Cycle. Nucleic Acids Res. 2017, 45 (20), 11525– 11534,  DOI: 10.1093/nar/gkx822
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    Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle
    Su'etsugu, Masayuki; Takada, Hiraku; Katayama, Tsutomu; Tsujimoto, Hiroko
    Nucleic Acids Research (2017), 45 (20), 11525-11534CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    Propagation of genetic information is a fundamental property of living organisms. Escherichia coli has a 4.6 Mb circular chromosome with a replication origin, oriC. While the oriC replication has been reconstituted in vitro more than 30 years ago, continuous repetition of the replication cycle has not yet been achieved. Here, we reconstituted the entire replication cycle with 14 purified enzymes (25 polypeptides) that catalyze initiation at oriC, bidirectional fork progression, Okazaki-fragment maturation and decatenation of the replicated circular products. Because decatenation provides covalently closed supercoiled monomers that are competent for the next round of replication initiation, the replication cycle repeats autonomously and continuously in an isothermal condition. This replication-cycle reaction (RCR) propagates ∼10 kb circular DNA exponentially as intact covalently closed mols., even from a single DNA mol., with a doubling time of ∼8 min and extremely high fidelity. Very large DNA up to 0.2 Mb is successfully propagated within 3 h. We further demonstrate a cell-free cloning in which RCR selectively propagates circular mols. constructed by a multi-fragment assembly reaction. Our results define the min. element necessary for the repetition of the chromosome-replication cycle, and also provide a powerful in vitro tool to generate large circular DNA mols. without relying on conventional biol. cloning.
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    Shimizu, Y.; Inoue, A.; Tomari, Y.; Suzuki, T.; Yokogawa, T.; Nishikawa, K.; Ueda, T. Cell-Free Translation Reconstituted with Purified Components. Nat. Biotechnol. 2001, 19 (8), 751– 755,  DOI: 10.1038/90802
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    Cell-free translation reconstituted with purified components
    Shimizu, Yoshihiro; Inoue, Akio; Tomari, Yukihide; Suzuki, Tsutomu; Yokogawa, Takashi; Nishikawa, Kazuya; Ueda, Takuya
    Nature Biotechnology (2001), 19 (8), 751-755CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
    We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 μg/mL/h in a batch mode without the need for any supplementary app. The protein products were easily purified within 1 h using affinity chromatog. to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor tRNA.
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    Drienovská, I.; Roelfes, G. Expanding the Enzyme Universe with Genetically Encoded Unnatural Amino Acids. Nature Catalysis 2020, 3 (3), 193– 202,  DOI: 10.1038/s41929-019-0410-8
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    Medina, E.; Yik, E. J.; Herdewijn, P.; Chaput, J. C. Functional Comparison of Laboratory-Evolved XNA Polymerases for Synthetic Biology. ACS Synth. Biol. 2021, 10 (6), 1429– 1437,  DOI: 10.1021/acssynbio.1c00048
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    Theobald, D. L. A Formal Test of the Theory of Universal Common Ancestry. Nature 2010, 465 (7295), 219– 222,  DOI: 10.1038/nature09014
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    Yang, Z.; Chen, F.; Alvarado, J. B.; Benner, S. A. Amplification, Mutation, and Sequencing of a Six-Letter Synthetic Genetic System. J. Am. Chem. Soc. 2011, 133 (38), 15105– 15112,  DOI: 10.1021/ja204910n
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    Amplification, mutation, and sequencing of a six-letter synthetic genetic system
    Yang, Zun-Yi; Chen, Fei; Alvarado, J. Brian; Benner, Steven A.
    Journal of the American Chemical Society (2011), 133 (38), 15105-15112CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The next goals in the development of a synthetic biol. that uses artificial genetic systems will require chem.-biol. combinations that allow the amplification of DNA contg. any no. of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technol. is needed to sequence artificial genetic DNA mols. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four std. nucleotides (G, A, C, and T) and two addnl. nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theor. cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.
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    Malyshev, D. A.; Dhami, K.; Lavergne, T.; Chen, T.; Dai, N.; Foster, J. M.; Corrêa, I. R., Jr; Romesberg, F. E. A Semi-Synthetic Organism with an Expanded Genetic Alphabet. Nature 2014, 509 (7500), 385– 388,  DOI: 10.1038/nature13314
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    A semi-synthetic organism with an expanded genetic alphabet
    Malyshev, Denis A.; Dhami, Kirandeep; Lavergne, Thomas; Chen, Tingjian; Dai, Nan; Foster, Jeremy M.; Correa, Ivan R.; Romesberg, Floyd E.
    Nature (London, United Kingdom) (2014), 509 (7500), 385-388CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA contg. the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid contg. d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
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    Rech, E. L.; Arber, W. Biodiversity as a Source for Synthetic Domestication of Useful Specific Traits. Ann. Appl. Biol. 2013, 162 (2), 141– 144,  DOI: 10.1111/aab.12013
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    Rech, E. Genomics and Synthetic Biology as a Viable Option to Intensify Sustainable Use of Biodiversity. Nat. Precedings 2011, 1– 1,  DOI: 10.1038/npre.2011.5759.1
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    Kiyama, H.; Kakizawa, S.; Sasajima, Y.; Tahara, Y. O.; Miyata, M. Reconstitution of a Minimal Motility System Based on Spiroplasma Swimming by Two Bacterial Actins in a Synthetic Minimal Bacterium. Sci. Adv. 2022, 8 (48), eabo7490,  DOI: 10.1126/sciadv.abo7490
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    Geiger, O.; Sanchez-Flores, A.; Padilla-Gomez, J.; Degli Esposti, M. Multiple Approaches of Cellular Metabolism Define the Bacterial Ancestry of Mitochondria. Sci. Adv. 2023, 9 (32), eadh0066,  DOI: 10.1126/sciadv.adh0066
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    Pinheiro, V. B.; Taylor, A. I.; Cozens, C.; Abramov, M.; Renders, M.; Zhang, S.; Chaput, J. C.; Wengel, J.; Peak-Chew, S.-Y.; McLaughlin, S. H.; Herdewijn, P.; Holliger, P. Synthetic Genetic Polymers Capable of Heredity and Evolution. Science 2012, 336 (6079), 341– 344,  DOI: 10.1126/science.1217622
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    Synthetic Genetic Polymers Capable of Heredity and Evolution
    Pinheiro, Vitor B.; Taylor, Alexander I.; Cozens, Christopher; Abramov, Mikhail; Renders, Marleen; Zhang, Su; Chaput, John C.; Wengel, Jesper; Peak-Chew, Sew-Yeu; McLaughlin, Stephen H.; Herdewijn, Piet; Holliger, Philipp
    Science (Washington, DC, United States) (2012), 336 (6079), 341-344CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Genetic information storage and processing rely on just two polymers, DNA and RNA, yet whether their role reflects evolutionary history or fundamental functional constraints is currently unknown. With the use of polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers based on simple nucleic acid architectures not found in nature [xeno-nucleic acids (XNAs)]. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and folding into defined structures. Thus, heredity and evolution, two hallmarks of life, are not limited to DNA and RNA but are likely to be emergent properties of polymers capable of information storage.
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    Hoshika, S.; Leal, N. A.; Kim, M.-J.; Kim, M.-S.; Karalkar, N. B.; Kim, H.-J.; Bates, A. M.; Watkins, N. E., Jr; SantaLucia, H. A.; Meyer, A. J.; DasGupta, S.; Piccirilli, J. A.; Ellington, A. D.; SantaLucia, J., Jr; Georgiadis, M. M.; Benner, S. A. Hachimoji DNA and RNA: A Genetic System with Eight Building Blocks. Science 2019, 363 (6429), 884– 887,  DOI: 10.1126/science.aat0971
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    Hachimoji DNA and RNA: A genetic system with eight building blocks
    Hoshika, Shuichi; Leal, Nicole A.; Kim, Myong-Jung; Kim, Myong-Sang; Karalkar, Nilesh B.; Kim, Hyo-Joong; Bates, Alison M.; Watkins, Norman E., Jr.; SantaLucia, Holly A.; Meyer, Adam J.; DasGupta, Saurja; Piccirilli, Joseph A.; Ellington, Andrew D.; SantaLucia, John, Jr.; Georgiadis, Millie M.; Benner, Steven A.
    Science (Washington, DC, United States) (2019), 363 (6429), 884-887CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodn. stability, and stereoregular building blocks that fit a Schroedinger aperiodic crystal. Measured thermodn. parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information d. of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of mol. structures that might support life, including life throughout the cosmos.
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  52. 52
    Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; Bridgland, A.; Meyer, C.; Kohl, S. A. A.; Ballard, A. J.; Cowie, A.; Romera-Paredes, B.; Nikolov, S.; Jain, R.; Adler, J.; Back, T.; Petersen, S.; Reiman, D.; Clancy, E.; Zielinski, M.; Steinegger, M.; Pacholska, M.; Berghammer, T.; Bodenstein, S.; Silver, D.; Vinyals, O.; Senior, A. W.; Kavukcuoglu, K.; Kohli, P.; Hassabis, D. Highly Accurate Protein Structure Prediction with AlphaFold. Nature 2021, 596 (7873), 583– 589,  DOI: 10.1038/s41586-021-03819-2
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    Highly accurate protein structure prediction with AlphaFold
    Jumper, John; Evans, Richard; Pritzel, Alexander; Green, Tim; Figurnov, Michael; Ronneberger, Olaf; Tunyasuvunakool, Kathryn; Bates, Russ; Zidek, Augustin; Potapenko, Anna; Bridgland, Alex; Meyer, Clemens; Kohl, Simon A. A.; Ballard, Andrew J.; Cowie, Andrew; Romera-Paredes, Bernardino; Nikolov, Stanislav; Jain, Rishub; Adler, Jonas; Back, Trevor; Petersen, Stig; Reiman, David; Clancy, Ellen; Zielinski, Michal; Steinegger, Martin; Pacholska, Michalina; Berghammer, Tamas; Bodenstein, Sebastian; Silver, David; Vinyals, Oriol; Senior, Andrew W.; Kavukcuoglu, Koray; Kohli, Pushmeet; Hassabis, Demis
    Nature (London, United Kingdom) (2021), 596 (7873), 583-589CODEN: NATUAS; ISSN:0028-0836. (Nature Portfolio)
    Proteins are essential to life, and understanding their structure can facilitate a mechanistic understanding of their function. Through an enormous exptl. effort, the structures of around 100,000 unique proteins have been detd., but this represents a small fraction of the billions of known protein sequences. Structural coverage is bottlenecked by the months to years of painstaking effort required to det. a single protein structure. Accurate computational approaches are needed to address this gap and to enable large-scale structural bioinformatics. Predicting the three-dimensional structure that a protein will adopt based solely on its amino acid sequence-the structure prediction component of the 'protein folding problem'-has been an important open research problem for more than 50 years. Despite recent progress, existing methods fall far short of at. accuracy, esp. when no homologous structure is available. Here we provide the first computational method that can regularly predict protein structures with at. accuracy even in cases in which no similar structure is known. We validated an entirely redesigned version of our neural network-based model, AlphaFold, in the challenging 14th Crit. Assessment of protein Structure Prediction (CASP14), demonstrating accuracy competitive with exptl. structures in a majority of cases and greatly outperforming other methods. Underpinning the latest version of AlphaFold is a novel machine learning approach that incorporates phys. and biol. knowledge about protein structure, leveraging multi-sequence alignments, into the design of the deep learning algorithm.
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    Lee, J. W.; Na, D.; Park, J. M.; Lee, J.; Choi, S.; Lee, S. Y. Systems Metabolic Engineering of Microorganisms for Natural and Non-Natural Chemicals. Nat. Chem. Biol. 2012, 8 (6), 536– 546,  DOI: 10.1038/nchembio.970
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    Systems metabolic engineering of microorganisms for natural and non-natural chemicals
    Lee, Jeong Wook; Na, Dokyun; Park, Jong Myoung; Lee, Joungmin; Choi, Sol; Lee, Sang Yup
    Nature Chemical Biology (2012), 8 (6), 536-546CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    A review. Growing concerns over limited fossil resources and assocd. environmental problems are motivating the development of sustainable processes for the prodn. of chems., fuels and materials from renewable resources. Metabolic engineering is a key enabling technol. for transforming microorganisms into efficient cell factories for these compds. Systems metabolic engineering, which incorporates the concepts and techniques of systems biol., synthetic biol. and evolutionary engineering at the systems level, offers a conceptual and technol. framework to speed the creation of new metabolic enzymes and pathways or the modification of existing pathways for the optimal prodn. of desired products. Here we discuss the general strategies of systems metabolic engineering and examples of its application and offer insights as to when and how each of the different strategies should be used. Finally, we highlight the limitations and challenges to be overcome for the systems metabolic engineering of microorganisms at more advanced levels.
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    Nielsen, J. Systems Biology of Metabolism. Annu. Rev. Biochem. 2017, 86, 245– 275,  DOI: 10.1146/annurev-biochem-061516-044757
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    Systems Biology of Metabolism
    Nielsen, Jens
    Annual Review of Biochemistry (2017), 86 (), 245-275CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
    Metab. is highly complex and involves thousands of different connected reactions; it is therefore necessary to use math. models for holistic studies. The use of math. models in biol. is referred to as systems biol. In this review, the principles of systems biol. are described, and two different types of math. models used for studying metab. are discussed: kinetic models and genome-scale metabolic models. The use of different omics technologies, including transcriptomics, proteomics, metabolomics, and fluxomics, for studying metab. is presented. Finally, the application of systems biol. for analyzing global regulatory structures, engineering the metab. of cell factories, and analyzing human diseases is discussed.
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    Choi, K. R.; Jang, W. D.; Yang, D.; Cho, J. S.; Park, D.; Lee, S. Y. Systems Metabolic Engineering Strategies: Integrating Systems and Synthetic Biology with Metabolic Engineering. Trends Biotechnol. 2019, 37 (8), 817– 837,  DOI: 10.1016/j.tibtech.2019.01.003
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    Systems Metabolic Engineering Strategies: Integrating Systems and Synthetic Biology with Metabolic Engineering
    Choi, Kyeong Rok; Jang, Woo Dae; Yang, Dongsoo; Cho, Jae Sung; Park, Dahyeon; Lee, Sang Yup
    Trends in Biotechnology (2019), 37 (8), 817-837CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
    A review. Metabolic engineering allows development of microbial strains efficiently producing chems. and materials, but it requires much time, effort, and cost to make the strains industrially competitive. Systems metabolic engineering, which integrates tools and strategies of systems biol., synthetic biol., and evolutionary engineering with traditional metabolic engineering, has recently been used to facilitate development of high-performance strains. The past decade has witnessed this interdisciplinary strategy continuously being improved toward the development of industrially competitive overproducer strains. In this article, current trends in systems metabolic engineering including tools and strategies are reviewed, focusing on recent developments in selection of host strains, metabolic pathway reconstruction, tolerance enhancement, and metabolic flux optimization. Also, future challenges and prospects are discussed.
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    Karim, A. S.; Dudley, Q. M.; Juminaga, A.; Yuan, Y.; Crowe, S. A.; Heggestad, J. T.; Garg, S.; Abdalla, T.; Grubbe, W. S.; Rasor, B. J.; Coar, D. N.; Torculas, M.; Krein, M.; Liew, F. E.; Quattlebaum, A.; Jensen, R. O.; Stuart, J. A.; Simpson, S. D.; Köpke, M.; Jewett, M. C. In Vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes for Cell Design. Nat. Chem. Biol. 2020, 16 (8), 912– 919,  DOI: 10.1038/s41589-020-0559-0
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    In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design
    Karim, Ashty S.; Dudley, Quentin M.; Juminaga, Alex; Yuan, Yongbo; Crowe, Samantha A.; Heggestad, Jacob T.; Garg, Shivani; Abdalla, Tanus; Grubbe, William S.; Rasor, Blake J.; Coar, David N.; Torculas, Maria; Krein, Michael; Liew, FungMin; Quattlebaum, Amy; Jensen, Rasmus O.; Stuart, Jeffrey A.; Simpson, Sean D.; Kopke, Michael; Jewett, Michael C.
    Nature Chemical Biology (2020), 16 (8), 912-919CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
    The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced nos. of validated genetic parts and a lack of high-throughput workflows. Here the authors describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. The authors demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate prodn. and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, the authors then scaled up the authors' highest-performing pathway, which improved in vivo 3-HB prodn. in Clostridium by 20-fold to 14.63 ± 0.48 g L-1. The authors expect iPROBE to accelerate design-build-test cycles for industrial biotechnol.
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    Dudley, Q. M.; Karim, A. S.; Nash, C. J.; Jewett, M. C. In Vitro Prototyping of Limonene Biosynthesis Using Cell-Free Protein Synthesis. Metab. Eng. 2020, 61, 251– 260,  DOI: 10.1016/j.ymben.2020.05.006
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    In vitro prototyping of limonene biosynthesis using cell-free protein synthesis
    Dudley, Quentin M.; Karim, Ashty S.; Nash, Connor J.; Jewett, Michael C.
    Metabolic Engineering (2020), 61 (), 251-260CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)
    Metabolic engineering of microorganisms to produce sustainable chems. has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-build-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large no. of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manuf. pathway enzymes in sep. reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in exts. from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concns. on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene prodn. in 24 h from 0.2 to 4.5 mM (23-610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.
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    Averesch, N. J. H.; Krömer, J. O. Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds-Present and Future Strain Construction Strategies. Front. Bioeng. Biotechnol. 2018, 6, 32,  DOI: 10.3389/fbioe.2018.00032
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    Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds-Present and Future Strain Construction Strategies
    Averesch Nils J H; Kromer Jens O
    Frontiers in bioengineering and biotechnology (2018), 6 (), 32 ISSN:2296-4185.
    The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Re)construction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations.
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    Jang, W. D.; Kim, G. B.; Lee, S. Y. An Interactive Metabolic Map of Bio-Based Chemicals. Trends Biotechnol. 2023, 41 (1), 10– 14,  DOI: 10.1016/j.tibtech.2022.07.013
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    Lai, H.-E.; Obled, A. M. C.; Chee, S. M.; Morgan, R. M.; Lynch, R.; Sharma, S. V.; Moore, S. J.; Polizzi, K. M.; Goss, R. J. M.; Freemont, P. S. GenoChemetic Strategy for Derivatization of the Violacein Natural Product Scaffold. ACS Chem. Biol. 2021, 16 (11), 2116– 2123,  DOI: 10.1021/acschembio.1c00483
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    GenoChemetic Strategy for Derivatization of the Violacein Natural Product Scaffold
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    ACS Chemical Biology (2021), 16 (11), 2116-2123CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Natural products and their analogs are often challenging to synthesize due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compd. diversity. Integrating synthetic biol. with synthetic chem. allows rapid access to much more diverse portfolios of xenobiotic compds., which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, six halogenated analogs of violacein or deoxyviolacein were generated, demonstrating the promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivs. were generated from 5-brominated violacein analogs via the Suzuki-Miyaura cross-coupling reaction directly using the crude ext. without prior purifn. Herein we demonstrate a flexible and rapid approach to access a diverse chem. space that can be applied to a wide range of natural product scaffolds.
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    Galanie, S.; Thodey, K.; Trenchard, I. J.; Filsinger Interrante, M.; Smolke, C. D. Complete Biosynthesis of Opioids in Yeast. Science 2015, 349 (6252), 1095– 1100,  DOI: 10.1126/science.aac9373
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    Complete biosynthesis of opioids in yeast
    Galanie, Stephanie; Thodey, Kate; Trenchard, Isis J.; Filsinger Interrante, Maria; Smolke, Christina D.
    Science (Washington, DC, United States) (2015), 349 (6252), 1095-1100CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Opioids are the primary drugs used in Western medicine for pain management and palliative care. Farming of opium poppies remains the sole source of these essential medicines, despite diverse market demands and uncertainty in crop yields due to weather, climate change, and pests. We engineered yeast to produce the selected opioid compds. thebaine and hydrocodone starting from sugar. All work was conducted in a lab. that is permitted and secured for work with controlled substances. We combined enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast. The resulting opioid biosynthesis strains required the expression of 21 (thebaine) and 23 (hydrocodone) enzyme activities from plants, mammals, bacteria, and yeast itself. This is a proof of principle, and major hurdles remain before optimization and scale-up could be achieved. Open discussions of options for governing this technol. are also needed in order to responsibly realize alternative supplies for these medically relevant compds.
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    Trenchard, I. J.; Siddiqui, M. S.; Thodey, K.; Smolke, C. D. De Novo Production of the Key Branch Point Benzylisoquinoline Alkaloid Reticuline in Yeast. Metab. Eng. 2015, 31, 74– 83,  DOI: 10.1016/j.ymben.2015.06.010
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    De novo production of the key branch point benzylisoquinoline alkaloid reticuline in yeast
    Trenchard, Isis J.; Siddiqui, Michael S.; Thodey, Kate; Smolke, Christina D.
    Metabolic Engineering (2015), 31 (), 74-83CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)
    Microbial biosynthesis for plant-based natural products, such as the benzylisoquinoline alkaloids (BIAs), has the potential to address limitations in plant-based supply of established drugs and make new mols. available for drug discovery. While yeast strains have been engineered to produce a variety of downstream BIAs including the opioids, these strains have relied on feeding an early BIA substrate. We describe the de novo synthesis of the major BIA branch point intermediate reticuline via norcoclaurine in Saccharomyces cerevisiae. Modifications were introduced into yeast central metab. to increase supply of the BIA precursor tyrosine, allowing us to achieve a 60-fold increase in prodn. of the early benzylisoquinoline scaffold from fed dopamine with no supply of exogenous tyrosine. Yeast strains further engineered to express a mammalian tyrosine hydroxylase, 4 mammalian tetrahydrobiopterin biosynthesis and recycling enzymes, and a bacterial DOPA decarboxylase produced norcoclaurine de novo. We further increased prodn. of early benzylisoquinoline scaffolds by 160-fold through introducing mutant tyrosine hydroxylase enzymes, an optimized plant norcoclaurine synthase variant, and optimizing culture conditions. Finally, we incorporated 5 addnl. plant enzymes (3 methyltransferases, a cytochrome P 450, and its reductase partner) to achieve de novo prodn. of the key branch point mol. reticuline with a titer of 19.2 μg/L. These strains and reconstructed pathways will serve as a platform for the biosynthesis of diverse natural and novel BIAs.
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    Chan, C. T. Y.; Lee, J. W.; Cameron, D. E.; Bashor, C. J.; Collins, J. J. Deadman” and “Passcode” Microbial Kill Switches for Bacterial Containment. Nat. Chem. Biol. 2016, 12 (2), 82– 86,  DOI: 10.1038/nchembio.1979
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    Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safeguard systems known as the 'Deadman' and 'Passcode' kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI-GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.
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    Caliando, B. J.; Voigt, C. A. Targeted DNA Degradation Using a CRISPR Device Stably Carried in the Host Genome. Nat. Commun. 2015, 6, 6989,  DOI: 10.1038/ncomms7989
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    Targeted DNA degradation using a CRISPR device stably carried in the host genome
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    Nature Communications (2015), 6 (), 6989CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    Once an engineered organism completes its task, it is useful to degrade the assocd. DNA to reduce environmental release and protect intellectual property. Here we present a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA. This enables engineered regions to be obscured when the cell enters a new environment. DNAi is based on type-IE CRISPR biochem. and a synthetic CRISPR array defines the DNA target(s). When the input is on, plasmid DNA is degraded 108-fold. When the genome is targeted, this causes cell death, reducing viable cells by a factor of 108. Further, the CRISPR nuclease can direct degrdn. to specific genomic regions (for example, engineered or inserted DNA), which could be used to complicate recovery and sequencing efforts. DNAi can be stably carried in an engineered organism, with no impact on cell growth, plasmid stability or DNAi inducibility even after passaging for >2 mo.
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    Lussier, F.; Staufer, O.; Platzman, I.; Spatz, J. P. Can Bottom-Up Synthetic Biology Generate Advanced Drug-Delivery Systems?. Trends Biotechnol. 2021, 39 (5), 445– 459,  DOI: 10.1016/j.tibtech.2020.08.002
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    Can Bottom-Up Synthetic Biology Generate Advanced Drug-Delivery Systems
    Lussier, Felix; Staufer, Oskar; Platzman, Ilia; Spatz, Joachim P.
    Trends in Biotechnology (2021), 39 (5), 445-459CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
    A review. Creating a magic bullet that can selectively kill cancer cells while sparing nearby healthy cells remains one of the most ambitious objectives in pharmacol. Nanomedicine, which relies on the use of nanotechnologies to fight disease, was envisaged to fulfill this coveted goal. Despite substantial progress, the structural complexity of therapeutic vehicles impedes their broad clin. application. Novel modular manufg. approaches for engineering programmable drug carriers may be able to overcome some fundamental limitations of nanomedicine. We discuss how bottom-up synthetic biol. principles, empowered by microfluidics, can palliate current drug carrier assembly limitations, and we demonstrate how such a magic bullet could be engineered from the bottom up to ultimately improve clin. outcomes for patients.
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    Staufer, O.; Dietrich, F.; Rimal, R.; Schröter, M.; Fabritz, S.; Boehm, H.; Singh, S.; Möller, M.; Platzman, I.; Spatz, J. P. Bottom-up Assembly of Biomedical Relevant Fully Synthetic Extracellular Vesicles. Sci. Adv. 2021, 7 (36), eabg6666,  DOI: 10.1126/sciadv.abg6666
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    Ghaemmaghamian, Z.; Zarghami, R.; Walker, G.; O’Reilly, E.; Ziaee, A. Stabilizing Vaccines via Drying: Quality by Design Considerations. Adv. Drug Delivery Rev. 2022, 187, 114313,  DOI: 10.1016/j.addr.2022.114313
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    Stabilizing vaccines via drying: Quality by design considerations
    Ghaemmaghamian, Zahra; Zarghami, Reza; Walker, Gavin; O'Reilly, Emmet; Ziaee, Ahmad
    Advanced Drug Delivery Reviews (2022), 187 (), 114313CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)
    A review. Pandemics and epidemics are continually challenging human beings' health and imposing major stresses on the societies particularly over the last few decades, when their frequency has increased significantly. Protecting humans from multiple diseases is best achieved through vaccination. However, vaccines thermal instability has always been a hurdle in their widespread application, esp. in less developed countries. Furthermore, insufficient vaccine processing capacity is also a major challenge for global vaccination programs. Continuous drying of vaccine formulations is one of the potential solns. to these challenges. This review highlights the challenges on implementing the continuous drying techniques for drying vaccines. The conventional drying methods, emerging technologies and their adaptation by biopharmaceutical industry are investigated considering the patented technologies for drying of vaccines. Moreover, the current progress in applying Quality by Design (QbD) in each of the drying techniques considering the crit. quality attributes (CQAs), crit. process parameters (CPPs) are comprehensively reviewed. An expert advice is presented on the required actions to be taken within the biopharmaceutical industry to move towards continuous stabilization of vaccines in the realm of QbD.
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    Adiga, R.; Al-Adhami, M.; Andar, A.; Borhani, S.; Brown, S.; Burgenson, D.; Cooper, M. A.; Deldari, S.; Frey, D. D.; Ge, X.; Guo, H.; Gurramkonda, C.; Jensen, P.; Kostov, Y.; LaCourse, W.; Liu, Y.; Moreira, A.; Mupparapu, K.; Peñalber-Johnstone, C.; Pilli, M.; Punshon-Smith, B.; Rao, A.; Rao, G.; Rauniyar, P.; Snovida, S.; Taurani, K.; Tilahun, D.; Tolosa, L.; Tolosa, M.; Tran, K.; Vattem, K.; Veeraraghavan, S.; Wagner, B.; Wilhide, J.; Wood, D. W.; Zuber, A. Point-of-Care Production of Therapeutic Proteins of Good-Manufacturing-Practice Quality. Nat. Biomed Eng. 2018, 2 (9), 675– 686,  DOI: 10.1038/s41551-018-0259-1
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    Point-of-care production of therapeutic proteins of good-manufacturing-practice quality
    Adiga, Rajani; Al-adhami, Mustafa; Andar, Abhay; Borhani, Shayan; Brown, Sheniqua; Burgenson, David; Cooper, Merideth A.; Deldari, Sevda; Frey, Douglas D.; Ge, Xudong; Guo, Hui; Gurramkonda, Chandrasekhar; Jensen, Penny; Kostov, Yordan; LaCourse, William; Liu, Yang; Moreira, Antonio; Mupparapu, KarunaSri; Penalber-Johnstone, Chariz; Pilli, Manohar; Punshon-Smith, Benjamin; Rao, Aniruddha; Rao, Govind; Rauniyar, Priyanka; Snovida, Sergei; Taurani, Kanika; Tilahun, Dagmawi; Tolosa, Leah; Tolosa, Michael; Tran, Kevin; Vattem, Krishna; Veeraraghavan, Sudha; Wagner, Brandon; Wilhide, Joshua; Wood, David W.; Zuber, Adil
    Nature Biomedical Engineering (2018), 2 (9), 675-686CODEN: NBEAB3; ISSN:2157-846X. (Nature Research)
    Manufg. technologies for biologics rely on large, centralized, good-manufg.-practice (GMP) prodn. facilities and on a cumbersome product-distribution network. Here, we report the development of an automated and portable medicines-on-demand device that enables consistent, small-scale GMP manufg. of therapeutic-grade biologics on a timescale of hours. The device couples the in vitro translation of target proteins from ribosomal DNA, using exts. from reconstituted lyophilized Chinese hamster ovary cells, with the continuous purifn. of the proteins. We used the device to reproducibly manuf. His-tagged granulocyte-colony stimulating factor, erythropoietin, glucose-binding protein and diphtheria toxoid DT5. Medicines-on-demand technol. may enable the rapid manufg. of biologics at the point of care.
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    Ginsburg, G. S.; Phillips, K. A. Precision Medicine: From Science To Value. Health Aff. 2018, 37 (5), 694– 701,  DOI: 10.1377/hlthaff.2017.1624
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    Agarwal, S.; Saha, S.; Balla, V. K.; Pal, A.; Barui, A.; Bodhak, S. Current Developments in 3D Bioprinting for Tissue and Organ Regeneration-A Review. Front. Mech. Eng. Chin 2020,  DOI: 10.3389/fmech.2020.589171
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    Ghosh, S.; Yi, H.-G. A Review on Bioinks and Their Application in Plant Bioprinting. Int. J. Bioprint 2022, 8 (4), 612,  DOI: 10.18063/ijb.v8i4.612
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    Santomartino, R.; Averesch, N. J. H.; Bhuiyan, M.; Cockell, C. S.; Colangelo, J.; Gumulya, Y.; Lehner, B.; Lopez-Ayala, I.; McMahon, S.; Mohanty, A.; Santa Maria, S. R.; Urbaniak, C.; Volger, R.; Yang, J.; Zea, L. Toward Sustainable Space Exploration: A Roadmap for Harnessing the Power of Microorganisms. Nat. Commun. 2023, 14 (1), 1391,  DOI: 10.1038/s41467-023-37070-2
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    Rothschild, L. J. Synthetic Biology Meets Bioprinting: Enabling Technologies for Humans on Mars (and Earth). Biochem. Soc. Trans. 2016, 44 (4), 1158– 1164,  DOI: 10.1042/BST20160067
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    Synthetic biology meets bioprinting: enabling technologies for humans on Mars (and Earth)
    Rothschild, Lynn J.
    Biochemical Society Transactions (2016), 44 (4), 1158-1164CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
    Human exploration off planet is severely limited by the cost of launching materials into space and by re-supply. Thus materials brought from Earth must be light, stable and reliable at destination. Using traditional approaches, a lunar or Mars base would require either transporting a hefty store of metals or heavy manufg. equipment and construction materials for in situ extn.; both would severely limit any other mission objectives. Long-term human space presence requires periodic replenishment, adding a massive cost overhead. Even robotic missions often sacrifice science goals for heavy radiation and thermal protection. Biol. has the potential to solve these problems because life can replicate and repair itself, and perform a wide variety of chem. reactions including making food, fuel and materials. Synthetic biol. enhances and expands life's evolved repertoire. Using organisms as feedstock, additive manufg. through bioprinting will make possible the dream of producing bespoke tools, food, smart fabrics and even replacement organs on demand. This new approach and the resulting novel products will enable human exploration and settlement on Mars, while providing new manufg. approaches for life on Earth.
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    Averesch, N. J. H.; Berliner, A. J.; Nangle, S. N.; Zezulka, S.; Vengerova, G. L.; Ho, D.; Casale, C. A.; Lehner, B. A. E.; Snyder, J. E.; Clark, K. B.; Dartnell, L. R.; Criddle, C. S.; Arkin, A. P. Microbial Biomanufacturing for Space-Exploration-What to Take and When to Make. Nat. Commun. 2023, 14 (1), 2311,  DOI: 10.1038/s41467-023-37910-1
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    Averesch, N. J. H. Choice of Microbial System for in-Situ Resource Utilization on Mars. Front. Astron. Space Sci. 2021,  DOI: 10.3389/fspas.2021.700370
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    Cockell, C. S. Bridging the Gap between Microbial Limits and Extremes in Space: Space Microbial Biotechnology in the next 15 Years. Microb. Biotechnol. 2022, 15 (1), 29– 41,  DOI: 10.1111/1751-7915.13927
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    Buddingh’, B. C.; van Hest, J. C. M. Artificial Cells: Synthetic Compartments with Life-like Functionality and Adaptivity. Acc. Chem. Res. 2017, 50 (4), 769– 777,  DOI: 10.1021/acs.accounts.6b00512
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    Artificial Cells: Synthetic Compartments with Life-like Functionality and Adaptivity
    Buddingh', Bastiaan C.; van Hest, Jan C. M.
    Accounts of Chemical Research (2017), 50 (4), 769-777CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. Cells are highly advanced microreactors that form the basis of all life. Their fascinating complexity has inspired scientists to create analogs from synthetic and natural components using a bottom-up approach. The ultimate goal here is to assemble a fully man-made cell that displays functionality and adaptivity as advanced as that found in nature, which will not only provide insight into the fundamental processes in natural cells but also pave the way for new applications of such artificial cells. In this Account, the authors highlight their recent work and that of others on the construction of artificial cells. First, the authors will introduce the key features that characterize a living system; next, the authors will discuss how these have been imitated in artificial cells. First, compartmentalization is crucial to sep. the inner chem. milieu from the external environment. Current state-of-the-art artificial cells comprise subcompartments to mimic the hierarchical architecture of eukaryotic cells and tissue. Furthermore, synthetic gene circuits have been used to encode genetic information that creates complex behavior like pulses or feedback. Addnl., artificial cells have to reproduce to maintain a population. Controlled growth and fission of synthetic compartments have been demonstrated, but the extensive regulation of cell division in nature is still unmatched. Here, the authors also point out important challenges the field needs to overcome to realize its full potential. As artificial cells integrate increasing orders of functionality, maintaining a supporting metab. that can regenerate key metabolites becomes crucial. Furthermore, life does not operate in isolation. Natural cells constantly sense their environment, exchange (chem.) signals, and can move toward a chemoattractant. Here, the authors specifically explore recent efforts to reproduce such adaptivity in artificial cells. For instance, synthetic compartments have been produced that can recruit proteins to the membrane upon an external stimulus or modulate their membrane compn. and permeability to control their interaction with the environment. A next step would be the communication of artificial cells with either bacteria or another artificial cell. Indeed, examples of such primitive chem. signaling are presented. Finally, motility is important for many organisms and has, therefore, also been pursued in synthetic systems. Synthetic compartments that were designed to move in a directed, controlled manner have been assembled, and directed movement toward a chem. attractant is among one of the most life-like directions currently under research. Although the bottom-up construction of an artificial cell that can be truly considered "alive" is still an ambitious goal, the recent work discussed in this Account shows that this is an active field with contributions from diverse disciplines like materials chem. and biochem. Notably, research during the past decade has already provided valuable insights into complex synthetic systems with life-like properties. In the future, artificial cells are thought to contribute to an increased understanding of processes in natural cells and provide opportunities to create smart, autonomous, cell-like materials.
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    Ichihashi, N. What Can We Learn from the Construction of in Vitro Replication Systems?. Ann. N.Y. Acad. Sci. 2019, 1447 (1), 144– 156,  DOI: 10.1111/nyas.14042
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    What can we learn from the construction of in vitro replication systems?
    Ichihashi Norikazu
    Annals of the New York Academy of Sciences (2019), 1447 (1), 144-156 ISSN:.
    Replication is a central function of living organisms. Several types of replication systems have been constructed in vitro from various molecules, including peptides, DNA, RNA, and proteins. In this review, I summarize the progress in the construction of replication systems over the past few decades and discuss what we can learn from their construction. I introduce various types of replication systems, supporting the feasibility of the spontaneous appearance of replication early in Earth's history. In the latter part of the review, I focus on parasitic replicators, one of the largest obstacles for sustainable replication. Compartmentalization is discussed as a possible solution.
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    Groaz, A.; Moghimianavval, H.; Tavella, F.; Giessen, T. W.; Vecchiarelli, A. G.; Yang, Q.; Liu, A. P. Engineering Spatiotemporal Organization and Dynamics in Synthetic Cells. WIREs Nanomed. Nanobiotechnol. 2021, 13 (3), e1685,  DOI: 10.1002/wnan.1685
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    Gaut, N. J.; Adamala, K. P. Reconstituting Natural Cell Elements in Synthetic Cells. Adv. Biol. 2021, 5 (3), e2000188,  DOI: 10.1002/adbi.202000188
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    Stal, L. J. Nitrogen Fixation in Cyanobacteria. In eLS; John Wiley & Sons, Ltd: Chichester, UK, 2015; pp 1– 9.  DOI: 10.1002/9780470015902.a0021159.pub2 .
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    Poirier, Y.; Antonenkov, V. D.; Glumoff, T.; Hiltunen, J. K. Peroxisomal β-oxidation─A Metabolic Pathway with Multiple Functions. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2006, 1763 (12), 1413– 1426,  DOI: 10.1016/j.bbamcr.2006.08.034
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    Peroxisomal β-oxidation-A metabolic pathway with multiple functions
    Poirier, Yves; Antonenkov, Vasily D.; Glumoff, Tuomo; Hiltunen, J. Kalervo
    Biochimica et Biophysica Acta, Molecular Cell Research (2006), 1763 (12), 1413-1426CODEN: BBAMCO; ISSN:0167-4889. (Elsevier Ltd.)
    A review. Fatty acid degrdn. in most organisms occurs primarily via the β-oxidn. cycle. In mammals, β-oxidn. occurs in both mitochondria and peroxisomes, whereas plants and most fungi harbor the β-oxidn. cycle only in the peroxisomes. Although several of the enzymes participating in this pathway in both organelles are similar, some distinct physiol. roles have been uncovered. Recent advances in the structural elucidation of numerous mammalian and yeast enzymes involved in β-oxidn. have shed light on the basis of the substrate specificity for several of them. Of particular interest is the structural organization and function of the type 1 and 2 multifunctional enzyme (MFE-1 and MFE-2), two enzymes evolutionarily distant yet catalyzing the same overall enzymic reactions but via opposite stereochem. New data on the physiol. roles of the various enzymes participating in β-oxidn. have been gathered through the anal. of knockout mutants in plants, yeast and animals, as well as by the use of polyhydroxyalkanoate synthesis from β-oxidn. intermediates as a tool to study carbon flux through the pathway. In plants, both forward and reverse genetics performed on the model plant Arabidopsis thaliana have revealed novel roles for β-oxidn. in the germination process that is independent of the generation of carbohydrates for growth, as well as in embryo and flower development, and the generation of the phytohormone indole-3-acetic acid and the signal mol. jasmonic acid.
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    Murat, D.; Byrne, M.; Komeili, A. Cell Biology of Prokaryotic Organelles. Cold Spring Harb. Perspect. Biol. 2010, 2 (10), a000422,  DOI: 10.1101/cshperspect.a000422
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    Nott, T. J.; Craggs, T. D.; Baldwin, A. J. Membraneless Organelles Can Melt Nucleic Acid Duplexes and Act as Biomolecular Filters. Nat. Chem. 2016, 8 (6), 569– 575,  DOI: 10.1038/nchem.2519
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    Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters
    Nott, Timothy J.; Craggs, Timothy D.; Baldwin, Andrew J.
    Nature Chemistry (2016), 8 (6), 569-575CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    Membraneless organelles are cellular compartments made from drops of liq. protein inside a cell. These compartments assemble via the phase sepn. of disordered regions of proteins in response to changes in the cellular environment and the cell cycle. Here we demonstrate that the solvent environment within the interior of these cellular bodies behaves more like an org. solvent than like water. One of the most-stable biol. structures known, the DNA double helix, can be melted once inside the liq. droplet, and simultaneously structures formed from regulatory single-stranded nucleic acids are stabilized. Moreover, proteins are shown to have a wide range of absorption or exclusion from these bodies, and can act as importers for otherwise-excluded nucleic acids, which suggests the existence of a protein-mediated trafficking system. A common strategy in org. chem. is to utilize different solvents to influence the behavior of mols. and reactions. These results reveal that cells have also evolved this capability by exploiting the interiors of membraneless organelles.
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    Lyon, A. S.; Peeples, W. B.; Rosen, M. K. A Framework for Understanding the Functions of Biomolecular Condensates across Scales. Nat. Rev. Mol. Cell Biol. 2021, 22 (3), 215– 235,  DOI: 10.1038/s41580-020-00303-z
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    A framework for understanding the functions of biomolecular condensates across scales
    Lyon, Andrew S.; Peeples, William B.; Rosen, Michael K.
    Nature Reviews Molecular Cell Biology (2021), 22 (3), 215-235CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)
    Abstr.: Biomol. condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing mols. that act in processes ranging from RNA metab. to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their phys. properties and regulation arise from mol. constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from mol. (modulating the rates of chem. reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochem. and cellular functions of biomol. condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biol.
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    Protter, D. S. W.; Parker, R. Principles and Properties of Stress Granules. Trends Cell Biol. 2016, 26 (9), 668– 679,  DOI: 10.1016/j.tcb.2016.05.004
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    Principles and Properties of Stress Granules
    Protter, David S. W.; Parker, Roy
    Trends in Cell Biology (2016), 26 (9), 668-679CODEN: TCBIEK; ISSN:0962-8924. (Elsevier Ltd.)
    A review. Stress granules are assemblies of untranslating messenger ribonucleoproteins (mRNPs) that form from mRNAs stalled in translation initiation. Stress granules form through interactions between mRNA-binding proteins that link together populations of mRNPs. Interactions promoting stress granule formation include conventional protein-protein interactions as well as interactions involving intrinsically disordered regions (IDRs) of proteins. Assembly and disassembly of stress granules are modulated by various post-translational modifications as well as numerous ATP-dependent RNP or protein remodeling complexes, illustrating that stress granules represent an active liq. wherein energy input maintains their dynamic state. Stress granule formation modulates the stress response, viral infection, and signaling pathways. Persistent or aberrant stress granule formation contributes to neurodegenerative disease and some cancers.
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    Roger, A. J.; Muñoz-Gómez, S. A.; Kamikawa, R. The Origin and Diversification of Mitochondria. Curr. Biol. 2017, 27 (21), R1177– R1192,  DOI: 10.1016/j.cub.2017.09.015
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    105
    The Origin and Diversification of Mitochondria
    Roger, Andrew J.; Munoz-Gomez, Sergio A.; Kamikawa, Ryoma
    Current Biology (2017), 27 (21), R1177-R1192CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
    A review. Mitochondria are best known for their role in the generation of ATP by aerobic respiration. Yet, research in the past half century has shown that they perform a much larger suite of functions and that these functions can vary substantially among diverse eukaryotic lineages. Despite this diversity, all mitochondria derive from a common ancestral organelle that originated from the integration of an endosymbiotic alphaproteobacterium into a host cell related to Asgard Archaea. The transition from endosymbiotic bacterium to permanent organelle entailed a massive no. of evolutionary changes including the origins of hundreds of new genes and a protein import system, insertion of membrane transporters, integration of metab. and reprodn., genome redn., endosymbiotic gene transfer, lateral gene transfer and the retargeting of proteins. These changes occurred incrementally as the endosymbiont and the host became integrated. Although many insights into this transition have been gained, controversy persists regarding the nature of the original endosymbiont, its initial interactions with the host and the timing of its integration relative to the origin of other features of eukaryote cells. Since the establishment of the organelle, proteins have been gained, lost, transferred and retargeted as mitochondria have specialized into the spectrum of functional types seen across the eukaryotic tree of life.
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  106. 106
    Mehta, A. P.; Supekova, L.; Chen, J.-H.; Pestonjamasp, K.; Webster, P.; Ko, Y.; Henderson, S. C.; McDermott, G.; Supek, F.; Schultz, P. G. Engineering Yeast Endosymbionts as a Step toward the Evolution of Mitochondria. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (46), 11796– 11801,  DOI: 10.1073/pnas.1813143115
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    106
    Engineering yeast endosymbionts as a step toward the evolution of mitochondria
    Mehta, Angad P.; Supekova, Lubica; Chen, Jian-Hua; Pestonjamasp, Kersi; Webster, Paul; Ko, Yeonjin; Henderson, Scott C.; McDermott, Gerry; Supek, Frantisek; Schultz, Peter G.
    Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (46), 11796-11801CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in an archaeal host cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between Escherichia coli and Saccharomyces cerevisiae. An ADP/ATP translocase-expressing E. coli provided ATP to a respiration-deficient cox2 yeast mutant and enabled growth of a yeast-E. coli chimera on a nonfermentable carbon source. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. Expression of several SNARE-like proteins in E. coli was also required, likely to block lysosomal degrdn. of intracellular bacteria. This chimeric system was stable for more than 40 doublings, and GFP-expressing E. coli endosymbionts could be obsd. in the yeast by fluorescence microscopy and X-ray tomog. This readily manipulated system should allow exptl. delineation of host-endosymbiont adaptations that occurred during evolution of the current, highly reduced mitochondrial genome.
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    Headen, D. M.; Aubry, G.; Lu, H.; García, A. J. Microfluidic-Based Generation of Size-Controlled, Biofunctionalized Synthetic Polymer Microgels for Cell Encapsulation. Adv. Mater. 2014, 26 (19), 3003– 3008,  DOI: 10.1002/adma.201304880
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    107
    Microfluidic-based generation of size-controlled, biofunctionalized synthetic polymer microgels for cell encapsulation
    Headen, Devon M.; Aubry, Guillaume; Lu, Hang; Garcia, Andres J.
    Advanced Materials (Weinheim, Germany) (2014), 26 (19), 3003-3008CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The high potential of synthetic hydrogel microencapsulation for cell and protein therapeutics has been limited by the lack of synthetic polymer systems with tunable capsule size, cytocompatible crosslinking reactions, rapid crosslinking rates, adequate biomol. permeability, and ease of functionalization with bioactive mols. (e.g., adhesive peptides). Using a synthetic hydrogel system with tunable network and crosslinking characteristics and a microfluidics encapsulation platform, we have created an integrated and robust strategy for microencapsulation of cells in which we can control capsule size and local cellular microenvironment. Addnl., microgel network structure can be tuned to optimize permeability of the capsule to mols. of various sizes. We have demonstrated, proof of concept with two different clin. relevant human cell types, but the versatility of this strategy will allow it to be tailored to fit diverse engineering applications.
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    Torre, P.; Keating, C. D.; Mansy, S. S. Multiphase Water-in-Oil Emulsion Droplets for Cell-Free Transcription-Translation. Langmuir 2014, 30 (20), 5695– 5699,  DOI: 10.1021/la404146g
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    Multiphase water-in-oil emulsion droplets for cell-free transcription-translation
    Torre, Paola; Keating, Christine D.; Mansy, Sheref S.
    Langmuir (2014), 30 (20), 5695-5699CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    The construction of genetically encoded cellular mimics in compartments contg. organized synthetic cytosols is desirable for the development of artificial cells. Phase sepd. aq. domains were placed within water-in-oil emulsion droplets in a manner compatible with transcription and translation machinery. Aq. two-phase and three-phase systems (ATPS and A3PS) were assembled with dextran, poly(ethylene glycol), and Ficoll. Aq. two-phase systems were capable of supporting the cell-free expression of protein within water droplets, whereas the aq. three-phase-based system did not give rise to detectable protein synthesis. The expressed protein preferentially partitioned to the dextran-enriched phase. The system could serve as a foundation for building cellular mimics with liq. organelles.
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    Zhang, Y.; Kojima, T.; Kim, G.-A.; McNerney, M. P.; Takayama, S.; Styczynski, M. P. Protocell Arrays for Simultaneous Detection of Diverse Analytes. Nat. Commun. 2021, 12 (1), 5724,  DOI: 10.1038/s41467-021-25989-3
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    Protocell arrays for simultaneous detection of diverse analytes
    Zhang, Yan; Kojima, Taisuke; Kim, Ge-Ah; McNerney, Monica P.; Takayama, Shuichi; Styczynski, Mark P.
    Nature Communications (2021), 12 (1), 5724CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Simultaneous detection of multiple analytes from a single sample (multiplexing), particularly when done at the point of need, can guide complex decision-making without increasing the required sample vol. or cost per test. Despite recent advances, multiplexed analyte sensing still typically faces the crit. limitation of measuring only one type of mol. (e.g., small mols. or nucleic acids) per assay platform. Here, we address this bottleneck with a customizable platform that integrates cell-free expression (CFE) with a polymer-based aq. two-phase system (ATPS), producing membrane-less protocells contg. transcription and translation machinery used for detection. We show that multiple protocells, each performing a distinct sensing reaction, can be arrayed in the same microwell to detect chem. diverse targets from the same sample. Furthermore, these protocell arrays are compatible with human biofluids, maintain function after lyophilization and rehydration, and can produce visually interpretable readouts, illustrating this platforms potential as a minimal-equipment, field-deployable, multi-analyte detection tool.
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    Allen, M. E.; Hindley, J. W.; Baxani, D. K.; Ces, O.; Elani, Y. Hydrogels as Functional Components in Artificial Cell Systems. Nat. Rev. Chem. 2022, 6 (8), 562– 578,  DOI: 10.1038/s41570-022-00404-7
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    110
    Hydrogels as functional components in artificial cell systems
    Allen, Matthew E.; Hindley, James W.; Baxani, Divesh K.; Ces, Oscar; Elani, Yuval
    Nature Reviews Chemistry (2022), 6 (8), 562-578CODEN: NRCAF7; ISSN:2397-3358. (Nature Portfolio)
    A review. Recent years have seen substantial efforts aimed at constructing artificial cells from various mol. components with the aim of mimicking the processes, behaviors and architectures found in biol. systems. Artificial cell development ultimately aims to produce model constructs that progress our understanding of biol., as well as forming the basis for functional bio-inspired devices that can be used in fields such as therapeutic delivery, biosensing, cell therapy and bioremediation. Typically, artificial cells rely on a bilayer membrane chassis and have fluid aq. interiors to mimic biol. cells. However, a desire to more accurately replicate the gel-like properties of intracellular and extracellular biol. environments has driven increasing efforts to build cell mimics based on hydrogels. This has enabled researchers to exploit some of the unique functional properties of hydrogels that have seen them deployed in fields such as tissue engineering, biomaterials and drug delivery. In this Review, we explore how hydrogels can be leveraged in the context of artificial cell development. We also discuss how hydrogels can potentially be incorporated within the next generation of artificial cells to engineer improved biol. mimics and functional microsystems.
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    Has, C.; Sunthar, P. A Comprehensive Review on Recent Preparation Techniques of Liposomes. J. Liposome Res. 2020, 30 (4), 336– 365,  DOI: 10.1080/08982104.2019.1668010
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    111
    A comprehensive review on recent preparation techniques of liposomes
    Has, C.; Sunthar, P.
    Journal of Liposome Research (2020), 30 (4), 336-365CODEN: JLREE7; ISSN:0898-2104. (Taylor & Francis Ltd.)
    A review Liposomes (or lipid vesicles) are a versatile platform as carriers for the delivery of the drugs and other macromols. into human and animal bodies. Though the method of using liposomes has been known since 1960s, and major developments and commercialization of liposomal formulations took place in the late nineties (or early part of this century), newer methods of liposome synthesis and drug encapsulation continue to be an active area of research. With the developments in related fields, such as electrohydrodynamics and microfluidics, and a growing understanding of the mechanisms of lipid assembly from colloidal and intermol. forces, liposome prepn. techniques have been enriched and more predictable than before. This has led to better methods that can also scale at an industrial prodn. level. In this review, we present several novel methods that were introduced over the last decade and compare their advantages over conventional methods. Researchers beginning to explore liposomal formulations will find this resource useful to give an overall direction for an appropriate choice of method. Where possible, we have also provided the known mechanisms behind the prepn. methods.
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  112. 112
    Luisi, P. L.; Allegretti, M.; Pereira de Souza, T.; Steiniger, F.; Fahr, A.; Stano, P. Spontaneous Protein Crowding in Liposomes: A New Vista for the Origin of Cellular Metabolism. Chembiochem 2010, 11 (14), 1989– 1992,  DOI: 10.1002/cbic.201000381
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    Michelon, M.; Huang, Y.; de la Torre, L. G.; Weitz, D. A.; Cunha, R. L. Single-Step Microfluidic Production of W/O/W Double Emulsions as Templates for β-Carotene-Loaded Giant Liposomes Formation. Chem. Eng. J. 2019, 366, 27– 32,  DOI: 10.1016/j.cej.2019.02.021
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    113
    Single-step microfluidic production of W/O/W double emulsions as templates for β-carotene-loaded giant liposomes formation
    Michelon, Mariano; Huang, Yuting; de la Torre, Lucimara Gaziola; Weitz, David A.; Cunha, Rosiane Lopes
    Chemical Engineering Journal (Amsterdam, Netherlands) (2019), 366 (), 27-32CODEN: CMEJAJ; ISSN:1385-8947. (Elsevier B.V.)
    We demonstrated the microfluidic prodn. of W/O/W double emulsion droplets aiming formation of β-carotene-incorporated giant liposomes for food and/or pharmaceutical applications. For this purpose, glass-capillary microfluidic devices were fabricated to create a truly three-dimensional flow aiming prodn. of giant unilamellar liposomes by solvent evapn. process after W/O/W double emulsion droplet templates formation. A great challenge of microfluidic prodn. of monodisperse and stable W/O/W double emulsion templates for this proposal is the replacement of org. solvents potentially toxic for phospholipids dissoln. Besides, the high cost of several semi-synthetic phospholipids commonly used for giant liposome formation remains as a major technol. challenge to be overcome. Thus, β-carotene-incorporated giant liposomes were generated using biocompatible solvents with low toxic potential (Et acetate and pentane) and non-purified soybean lecithin - a food-grade phospholipid mixt. with low cost - by dewetting and evapn. of the solvents forming the oily intermediate phase of W/O/W double emulsion droplet templates. Our results showed monodisperse β-carotene-loaded giant liposomes with diam. ranging between 100 μm and 180 μm and a stability of approx. 7 days. In this way, a single-step microfluidic process with highly accurate control of size distribution was developed. This microfluidic process proposed is potentially useful for a broad range of applications in protection and delivery of active compds.
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  114. 114
    Elani, Y.; Trantidou, T.; Wylie, D.; Dekker, L.; Polizzi, K.; Law, R. V.; Ces, O. Constructing Vesicle-Based Artificial Cells with Embedded Living Cells as Organelle-like Modules. Sci. Rep. 2018, 8 (1), 4564,  DOI: 10.1038/s41598-018-22263-3
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    114
    Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules
    Elani Yuval; Trantidou Tatiana; Wylie Douglas; Law Robert V; Ces Oscar; Elani Yuval; Wylie Douglas; Ces Oscar; Dekker Linda; Polizzi Karen
    Scientific reports (2018), 8 (1), 4564 ISSN:.
    There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.
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    Tayeb, H. H.; Sainsbury, F. Nanoemulsions in Drug Delivery: Formulation to Medical Application. Nanomedicine 2018, 13 (19), 2507– 2525,  DOI: 10.2217/nnm-2018-0088
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    115
    Nanoemulsions in drug delivery: formulation to medical application
    Tayeb, Hossam H.; Sainsbury, Frank
    Nanomedicine (London, United Kingdom) (2018), 13 (19), 2507-2525CODEN: NLUKAC; ISSN:1748-6963. (Future Medicine Ltd.)
    Nanoscale oil-in-water emulsions (NEs), heterogeneous systems of two immiscible liqs. stabilized by emulsifiers or surfactants, show great potential in medical applications because of their attractive characteristics for drug delivery. NEs have been explored as therapeutic carriers for hydrophobic compds. via various routes of administration. NEs provide opportunities to improve drug delivery via alternative administration routes. However, deep understanding of the NE manufg. and functionalization fundamentals, and how they relate to the choice of administration route and pharmacol. profile is still needed to ease the clin. translation of NEs. Here, we review the diversity of medical applications for NEs and how that governs their formulation, route of administration, and the emergence of increasing sophistication in NE design for specific application.
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  116. 116
    Vogele, K.; Frank, T.; Gasser, L.; Goetzfried, M. A.; Hackl, M. W.; Sieber, S. A.; Simmel, F. C.; Pirzer, T. Towards Synthetic Cells Using Peptide-Based Reaction Compartments. Nat. Commun. 2018, 9 (1), 3862,  DOI: 10.1038/s41467-018-06379-8
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    116
    Towards synthetic cells using peptide-based reaction compartments
    Vogele Kilian; Frank Thomas; Gasser Lukas; Goetzfried Marisa A; Simmel Friedrich C; Pirzer Tobias; Hackl Mathias W; Sieber Stephan A; Simmel Friedrich C
    Nature communications (2018), 9 (1), 3862 ISSN:.
    Membrane compartmentalization and growth are central aspects of living cells, and are thus encoded in every cell's genome. For the creation of artificial cellular systems, genetic information and production of membrane building blocks will need to be coupled in a similar manner. However, natural biochemical reaction networks and membrane building blocks are notoriously difficult to implement in vitro. Here, we utilized amphiphilic elastin-like peptides (ELP) to create self-assembled vesicular structures of about 200 nm diameter. In order to genetically encode the growth of these vesicles, we encapsulate a cell-free transcription-translation system together with the DNA template inside the peptide vesicles. We show in vesiculo production of a functioning fluorescent RNA aptamer and a fluorescent protein. Furthermore, we implement in situ expression of the membrane peptide itself and finally demonstrate autonomous vesicle growth due to the incorporation of this ELP into the membrane.
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    Mushnoori, S.; Lu, C. Y.; Schmidt, K.; Zang, E.; Dutt, M. Peptide-Based Vesicles and Droplets: A Review. J. Phys.: Condens. Matter 2020, 33 (5), 053002,  DOI: 10.1088/1361-648X/abb995
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    Sharma, B.; Ma, Y.; Hiraki, H. L.; Baker, B. M.; Ferguson, A. L.; Liu, A. P. Facile Formation of Giant Elastin-like Polypeptide Vesicles as Synthetic Cells. Chem. Commun. 2021, 57 (97), 13202– 13205,  DOI: 10.1039/D1CC05579H
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    118
    Facile formation of giant elastin-like polypeptide vesicles as synthetic cells
    Sharma, Bineet; Ma, Yutao; Hiraki, Harrison L.; Baker, Brendon M.; Ferguson, Andrew L.; Liu, Allen P.
    Chemical Communications (Cambridge, United Kingdom) (2021), 57 (97), 13202-13205CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    We demonstrate the facile and robust generation of giant peptide vesicles by using an emulsion transfer method. These robust vesicles can sustain chem. and phys. stresses. The peptide vesicles can host cell-free expression reactions by encapsulating essential ingredients. We show the incorporation of another cell-free expressed elastin-like polypeptide into the existing membrane of the peptide vesicles.
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  119. 119
    Vieregg, J. R.; Lueckheide, M.; Marciel, A. B.; Leon, L.; Bologna, A. J.; Rivera, J. R.; Tirrell, M. V. Oligonucleotide-Peptide Complexes: Phase Control by Hybridization. J. Am. Chem. Soc. 2018, 140 (5), 1632– 1638,  DOI: 10.1021/jacs.7b03567
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    119
    Oligonucleotide-peptide complexes: Phase control by hybridization
    Vieregg, Jeffrey R.; Lueckheide, Michael; Marciel, Amanda B.; Leon, Lorraine; Bologna, Alex J.; Rivera, Josean Reyes; Tirrell, Matthew V.
    Journal of the American Chemical Society (2018), 140 (5), 1632-1638CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    When oppositely charged polymers are mixed, counterion release drives phase sepn.; understanding this process is a key unsolved problem in polymer science and biophys. chem., particularly for nucleic acids, polyanions whose biol. functions are intimately related to their high charge d. In the cell, complexation by basic proteins condenses DNA into chromatin, and membraneless organelles formed by liq.-liq. phase sepn. of RNA and proteins perform vital functions and have been linked to disease. Electrostatic interactions are also the primary method used for assembly of nanoparticles to deliver therapeutic nucleic acids into cells. This work describes complexation expts. with oligonucleotides and cationic peptides spanning a wide range of polymer lengths, concns., and structures, including RNA and methylphosphonate backbones. We find that the phase of the complexes is controlled by the hybridization state of the nucleic acid, with double-stranded nucleic acids forming solid ppts. while single-stranded oligonucleotides form liq. coacervates, apparently due to their lower charge d. Adding salt "melts" ppts. into coacervates, and oligonucleotides in coacervates remain competent for sequence-specific hybridization and phase change, suggesting the possibility of environmentally responsive complexes and nanoparticles for therapeutic or sensing applications.
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  120. 120
    Fraccia, T. P.; Jia, T. Z. Liquid Crystal Coacervates Composed of Short Double-Stranded DNA and Cationic Peptides. ACS Nano 2020, 14 (11), 15071– 15082,  DOI: 10.1021/acsnano.0c05083
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    120
    Liquid Crystal Coacervates Composed of Short Double-Stranded DNA and Cationic Peptides
    Fraccia, Tommaso P.; Jia, Tony Z.
    ACS Nano (2020), 14 (11), 15071-15082CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Phase sepn. of nucleic acids and proteins is a ubiquitous phenomenon regulating subcellular compartment structure and function. While complex coacervation of flexible single-stranded nucleic acids is broadly investigated, coacervation of double-stranded DNA (dsDNA) is less studied because of its propensity to generate solid ppts. Here, we reverse this perspective by showing that short dsDNA and poly-L-lysine coacervates can escape pptn. while displaying a surprisingly complex phase diagram, including the full set of liq. crystal (LC) mesophases obsd. to date in bulk dsDNA. Short dsDNA supramol. aggregation and packing in the dense coacervate phase are the main parameters regulating the global LC-coacervate phase behavior. LC-coacervate structure was characterized upon variations in temp. and monovalent salt, DNA, and peptide concns., which allow continuous reversible transitions between all accessible phases. A deeper understanding of LC-coacervates can gain insights to decipher structures and phase transition mechanisms within biomol. condensates, to design stimuli-responsive multiphase synthetic compartments with different degrees of order and to exploit self-assembly driven cooperative prebiotic evolution of nucleic acids and peptides.
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  121. 121
    Aumiller, W. M., Jr; Pir Cakmak, F.; Davis, B. W.; Keating, C. D. RNA-Based Coacervates as a Model for Membraneless Organelles: Formation, Properties, and Interfacial Liposome Assembly. Langmuir 2016, 32 (39), 10042– 10053,  DOI: 10.1021/acs.langmuir.6b02499
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    121
    RNA-Based Coacervates as a Model for Membraneless Organelles: Formation, Properties, and Interfacial Liposome Assembly
    Aumiller, William M.; Pir Cakmak, Fatma; Davis, Bradley W.; Keating, Christine D.
    Langmuir (2016), 32 (39), 10042-10053CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Liq.-liq. phase sepn. is responsible for the formation of P granules, nucleoli, and other membraneless subcellular organelles composed of RNA and proteins. Efforts to understand the phys. basis of liq. organelle formation have thus far focused on intrinsically disordered proteins (IDPs) as major components that dictate occurrence and properties. Here, the authors show that complex coacervates composed of low complexity RNA [polyuridylic acid, poly(U)] and short polyamines (spermine and spermidine) share many features of IDP-based coacervates. Poly(U)/polyamine coacervates compartmentalized biomols. (peptides, oligonucleotides) in a sequence- and length- dependent manner. These solutes retained mobility within the coacervate droplets, as demonstrated by rapid recovery from photobleaching. Coacervation was reversible with changes in soln. temp. due to changes in the poly(U) structure that impacted its interactions with polyamines. The authors further demonstrated that lipid vesicles assembled at the droplet interface without impeding RNA entry/egress. These vesicles remained intact at the interface and could be released upon temp.-induced droplet dissoln.
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  122. 122
    Rimoli, M. G.; Rabaioli, M. R.; Melisi, D.; Curcio, A.; Mondello, S.; Mirabelli, R.; Abignente, E. Synthetic Zeolites as a New Tool for Drug Delivery. J. Biomed. Mater. Res., Part A 2008, 87 (1), 156– 164,  DOI: 10.1002/jbm.a.31763
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    122
    Synthetic zeolites as a new tool for drug delivery
    Rimoli, Maria G.; Rabaioli, Maria R.; Melisi, Daniela; Curcio, Annalisa; Mondello, Sandro; Mirabelli, Rosella; Abignente, Enrico
    Journal of Biomedical Materials Research, Part A (2008), 87A (1), 156-164CODEN: JBMRCH; ISSN:1549-3296. (John Wiley & Sons, Inc.)
    Synthetic zeolites were studied in order to investigate their ability to encapsulate and to release drugs. In particular, a zeolite X and a zeolitic product obtained from a cocrystn. of zeolite X and zeolite A were examd. These materials were characterized by chem. analyses (ICP-AES), x-ray diffraction, nitrogen adsorption isotherm, SEM, laser diffraction, and IR spectroscopy. Since ketoprofen was chosen as a model drug for the formulation of controlled-release dosage forms, it was encapsulated into these two types of synthetic zeolites by a soaking procedure. Drug-loaded matrixes were then characterized for entrapped drug amt. and thermogravimetric behavior. In both types of activated zeolites, the total amt. of ketoprofen (800 mg) was encapsulated in 2 g of matrix. By using HPLC measurements, ketoprofen release studies were done at different pH conditions so as to mimic gastrointestinal fluids. The absence of release in acid conditions and a double phased release, at two different pH values (5 and 6.8), suggest that after activation these materials offer good potential for a modified release delivery system of ketoprofen.
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    Hyman, A. A.; Weber, C. A.; Jülicher, F. Liquid-Liquid Phase Separation in Biology. Annu. Rev. Cell Dev. Biol. 2014, 30, 39– 58,  DOI: 10.1146/annurev-cellbio-100913-013325
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    Liquid-liquid phase separation in biology
    Hyman, Anthony A.; Weber, Christoph A.; Juelicher, Frank
    Annual Review of Cell and Developmental Biology (2014), 30 (), 39-58CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews)
    A review. Cells organize many of their biochem. reactions in non-membrane compartments. Recent evidence showed that many of these compartments are liqs. that form by phase sepn. from the cytoplasm. Here the basic phys. concepts necessary to understand the consequences of liq.-like states for biol. functions are discussed.
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    Junge, F.; Haberstock, S.; Roos, C.; Stefer, S.; Proverbio, D.; Dötsch, V.; Bernhard, F. Advances in Cell-Free Protein Synthesis for the Functional and Structural Analysis of Membrane Proteins. N. Biotechnol. 2011, 28 (3), 262– 271,  DOI: 10.1016/j.nbt.2010.07.002
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    Lee, K. Y.; Park, S.-J.; Lee, K. A.; Kim, S.-H.; Kim, H.; Meroz, Y.; Mahadevan, L.; Jung, K.-H.; Ahn, T. K.; Parker, K. K.; Shin, K. Photosynthetic Artificial Organelles Sustain and Control ATP-Dependent Reactions in a Protocellular System. Nat. Biotechnol. 2018, 36 (6), 530– 535,  DOI: 10.1038/nbt.4140
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    Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system
    Lee, Keel Yong; Park, Sung-Jin; Lee, Keon Ah; Kim, Se-Hwan; Kim, Heeyeon; Meroz, Yasmine; Mahadevan, L.; Jung, Kwang-Hwan; Ahn, Tae Kyu; Parker, Kevin Kit; Shin, Kwanwoo
    Nature Biotechnology (2018), 36 (6), 530-535CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
    Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymn., with the latter altering outer vesicle morphol. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.
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    Jewett, M. C.; Swartz, J. R. Mimicking the Escherichia Coli Cytoplasmic Environment Activates Long-Lived and Efficient Cell-Free Protein Synthesis. Biotechnol. Bioeng. 2004, 86 (1), 19– 26,  DOI: 10.1002/bit.20026
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    Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis
    Jewett, Michael C.; Swartz, James R.
    Biotechnology and Bioengineering (2004), 86 (1), 19-26CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
    Cell-free translation systems generally utilize high-energy phosphate compds. to regenerate the ATP necessary to drive protein synthesis. This hampers the widespread use and practical implementation of this technol. in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change. To address these problems, a cell-free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein. The "Cytomim" system, synthesizes chloramphenicol acetyl-transferase (CAT) for up to 6 h in a batch reaction to yield 700 μg/mL of protein. By more closely replicating the physiol. conditions of the cytoplasm of Escherichia coli, the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addn., or the need for expensive high-energy phosphate compds.
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    Caschera, F.; Noireaux, V. Synthesis of 2.3 Mg/mL of Protein with an All Escherichia Coli Cell-Free Transcription-Translation System. Biochimie 2014, 99, 162– 168,  DOI: 10.1016/j.biochi.2013.11.025
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    Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system
    Caschera, Filippo; Noireaux, Vincent
    Biochimie (2014), 99 (), 162-168CODEN: BICMBE; ISSN:0300-9084. (Elsevier Masson SAS)
    Cell-free protein synthesis is becoming a useful technique for synthetic biol. As more applications are developed, the demand for novel and more powerful in vitro expression systems is increasing. In this work, an all Escherichia coli cell-free system, that uses the endogenous transcription and translation mol. machineries, is optimized to synthesize up to 2.3 mg/mL of a reporter protein in batch mode reactions. A new metab. based on maltose allows recycling of inorg. phosphate through its incorporation into newly available glucose mols., which are processed through the glycolytic pathway to produce more ATP. As a result, the ATP regeneration is more efficient and cell-free protein synthesis lasts up to 10 h. Using a com. E. coli strain, we show for the first time that more than 2 mg/mL of protein can be synthesized in run-off cell-free transcription-translation reactions by optimizing the energy regeneration and waste products recycling. This work suggests that endogenous enzymes present in the cytoplasmic ext. can be used to implement new metabolic pathways for increasing protein yields. This system is the new basis of a cell-free gene expression platform used to construct and to characterize complex biochem. processes in vitro such as gene circuits.
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    Garamella, J.; Marshall, R.; Rustad, M.; Noireaux, V. The All E. Coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology. ACS Synth. Biol. 2016, 5 (4), 344– 355,  DOI: 10.1021/acssynbio.5b00296
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    128
    The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology
    Garamella, Jonathan; Marshall, Ryan; Rustad, Mark; Noireaux, Vincent
    ACS Synthetic Biology (2016), 5 (4), 344-355CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic ext., thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metab. for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on av., the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. MRNAs and protein degrdn., resp. tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biol. systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.
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    Kim, D.-M.; Swartz, J. R. Efficient Production of a Bioactive, Multiple Disulfide-Bonded Protein Using Modified Extracts of Escherichia Coli. Biotechnol. Bioeng. 2004, 85 (2), 122– 129,  DOI: 10.1002/bit.10865
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    Jeong, S.; Nguyen, H. T.; Kim, C. H.; Ly, M. N.; Shin, K. Toward Artificial Cells: Novel Advances in Energy Conversion and Cellular Motility. Adv. Funct. Mater. 2020, 30 (11), 1907182,  DOI: 10.1002/adfm.201907182
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    Toward Artificial Cells: Novel Advances in Energy Conversion and Cellular Motility
    Jeong, Sungwoo; Nguyen, Huong Thanh; Kim, Chang Ho; Ly, Mai Nguyet; Shin, Kwanwoo
    Advanced Functional Materials (2020), 30 (11), 1907182CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. The demand to discover every single cellular component has been continuously increasing along with the development of biol. techniques. The bottom-up approach to construct a cell-mimicking system from well-defined and tunable compns. is accelerating, with the ultimate goal of comprehending a biol. cell. From among the available techniques, the artificial cell has been increasingly recognized as one of the most powerful tools for building a cell-like system from scratch. This review summarizes the development of artificial cells, from a pure giant unilamellar vesicle (GUV) to a controllable, self-fueled proteoliposome, both of which are highly compartmentalized. The basic components of an artificial cell, as well as the optimal conditions required for successful, reproducible GUV formation and protein reconstitution, are discussed. Most importantly, progress in studying the metabolic reactions in and the motility of a reconstituted artificial cell are the main focus of the review. The ability to perform a complicated reaction cascade in a controllable manner is highlighted as a promising perspective to obtaining an autonomous and movable GUV.
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    Hanczyc, M. M.; Fujikawa, S. M.; Szostak, J. W. Experimental Models of Primitive Cellular Compartments: Encapsulation, Growth, and Division. Science 2003, 302 (5645), 618– 622,  DOI: 10.1126/science.1089904
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    131
    Experimental Models of Primitive Cellular Compartments: Encapsulation, Growth, and Division
    Hanczyc, Martin M.; Fujikawa, Shelly M.; Szostak, Jack W.
    Science (Washington, DC, United States) (2003), 302 (5645), 618-622CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The clay montmorillonite is known to catalyze the polymn. of RNA from activated ribonucleotides. Here we report that montmorillonite accelerates the spontaneous conversion of fatty acid micelles into vesicles. Clay particles often become encapsulated in these vesicles, thus providing a pathway for the prebiotic encapsulation of catalytically active surfaces within membrane vesicles. In addn., RNA adsorbed to clay can be encapsulated within vesicles. Once formed, such vesicles can grow by incorporating fatty acid supplied as micelles and can divide without diln. of their contents by extrusion through small pores. These processes mediate vesicle replication through cycles of growth and division. The formation, growth, and division of the earliest cells may have occurred in response to similar interactions with mineral particles and inputs of material and energy.
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    Kretschmer, S.; Ganzinger, K. A.; Franquelim, H. G.; Schwille, P. Synthetic Cell Division via Membrane-Transforming Molecular Assemblies. BMC Biol. 2019, 17 (1), 43,  DOI: 10.1186/s12915-019-0665-1
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    Synthetic cell division via membrane-transforming molecular assemblies
    Kretschmer Simon; Ganzinger Kristina A; Franquelim Henri G; Schwille Petra; Kretschmer Simon; Ganzinger Kristina A
    BMC biology (2019), 17 (1), 43 ISSN:.
    Reproduction, i.e. the ability to produce new individuals from a parent organism, is a hallmark of living matter. Even the simplest forms of reproduction require cell division: attempts to create a designer cell therefore should include a synthetic cell division machinery. In this review, we will illustrate how nature solves this task, describing membrane remodelling processes in general and focusing on bacterial cell division in particular. We discuss recent progress made in their in vitro reconstitution, identify open challenges, and suggest how purely synthetic building blocks could provide an additional and attractive route to creating artificial cell division machineries.
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    Anzai, K.; Yoshida, M.; Kirino, Y. Change in Intravesicular Volume of Liposomes by Freeze-Thaw Treatment as Studied by the ESR Stopped-Flow Technique. Biochimica et Biophysica Acta (BBA) - Biomembranes 1990, 1021 (1), 21– 26,  DOI: 10.1016/0005-2736(90)90378-2
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    van der Valk, T.; Pečnerová, P.; Díez-Del-Molino, D.; Bergström, A.; Oppenheimer, J.; Hartmann, S.; Xenikoudakis, G.; Thomas, J. A.; Dehasque, M.; Sağlıcan, E.; Fidan, F. R.; Barnes, I.; Liu, S.; Somel, M.; Heintzman, P. D.; Nikolskiy, P.; Shapiro, B.; Skoglund, P.; Hofreiter, M.; Lister, A. M.; Götherström, A.; Dalén, L. Million-Year-Old DNA Sheds Light on the Genomic History of Mammoths. Nature 2021, 591 (7849), 265– 269,  DOI: 10.1038/s41586-021-03224-9
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    Million-year-old DNA sheds light on the genomic history of mammoths
    van der Valk, Tom; Pecnerova, Patricia; Diez-del-Molino, David; Bergstroem, Anders; Oppenheimer, Jonas; Hartmann, Stefanie; Xenikoudakis, Georgios; Thomas, Jessica A.; Dehasque, Marianne; Saglican, Ekin; Fidan, Fatma Rabia; Barnes, Ian; Liu, Shanlin; Somel, Mehmet; Heintzman, Peter D.; Nikolskiy, Pavel; Shapiro, Beth; Skoglund, Pontus; Hofreiter, Michael; Lister, Adrian M.; Goetherstroem, Anders; Dalen, Love
    Nature (London, United Kingdom) (2021), 591 (7849), 265-269CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Temporal genomic data hold great potential for studying evolutionary processes such as speciation. However, sampling across speciation events would, in many cases, require genomic time series that stretch well back into the Early Pleistocene subepoch. Although theor. models suggest that DNA should survive on this timescale1, the oldest genomic data recovered so far are from a horse specimen dated to 780-560 thousand years ago2. Here we report the recovery of genome-wide data from three mammoth specimens dating to the Early and Middle Pleistocene subepochs, two of which are more than one million years old. We find that two distinct mammoth lineages were present in eastern Siberia during the Early Pleistocene. One of these lineages gave rise to the woolly mammoth and the other represents a previously unrecognized lineage that was ancestral to the first mammoths to colonize North America. Our analyses reveal that the Columbian mammoth of North America traces its ancestry to a Middle Pleistocene hybridization between these two lineages, with roughly equal admixt. proportions. Finally, we show that the majority of protein-coding changes assocd. with cold adaptation in woolly mammoths were already present one million years ago. These findings highlight the potential of deep-time palaeogenomics to expand our understanding of speciation and long-term adaptive evolution.
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    Li, Z.; Deutscher, M. P. Analyzing the Decay of Stable RNAs in E. Coli. Methods Enzymol. 2008, 447, 31– 45,  DOI: 10.1016/S0076-6879(08)02202-7
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    Chan, L. Y.; Mugler, C. F.; Heinrich, S.; Vallotton, P.; Weis, K. Non-Invasive Measurement of mRNA Decay Reveals Translation Initiation as the Major Determinant of mRNA Stability. eLife 2018,  DOI: 10.7554/eLife.32536
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    Paul, N.; Joyce, G. F. A Self-Replicating Ligase Ribozyme. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (20), 12733– 12740,  DOI: 10.1073/pnas.202471099
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    Robertson, M. P.; Joyce, G. F. Highly Efficient Self-Replicating RNA Enzymes. Chem. Biol. 2014, 21 (2), 238– 245,  DOI: 10.1016/j.chembiol.2013.12.004
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    Highly Efficient Self-Replicating RNA Enzymes
    Robertson, Michael P.; Joyce, Gerald F.
    Chemistry & Biology (Oxford, United Kingdom) (2014), 21 (2), 238-245CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
    An RNA enzyme has been developed that catalyzes the joining of oligonucleotide substrates to form addnl. copies of itself, undergoing self-replication with exponential growth. The enzyme also can cross-replicate with a partner enzyme, resulting in their mutual exponential growth and enabling self-sustained Darwinian evolution. The opportunity for inventive evolution within this synthetic genetic system depends on the diversity of the evolving population, which is limited by the catalytic efficiency of the enzyme. Directed evolution was used to improve the efficiency of the enzyme and increase its exponential growth rate to 0.14 min-1, corresponding to a doubling time of 5 min. This is close to the limit of 0.21 min-1 imposed by the rate of product release, but sufficient to enable more than 80 logs of growth per day.
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    Chen, I. A.; Salehi-Ashtiani, K.; Szostak, J. W. RNA Catalysis in Model Protocell Vesicles. J. Am. Chem. Soc. 2005, 127 (38), 13213– 13219,  DOI: 10.1021/ja051784p
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    RNA Catalysis in Model Protocell Vesicles
    Chen, Irene A.; Salehi-Ashtiani, Kourosh; Szostak, Jack W.
    Journal of the American Chemical Society (2005), 127 (38), 13213-13219CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    We are engaged in a long-term effort to synthesize chem. systems capable of Darwinian evolution, based on the encapsulation of self-replicating nucleic acids in self-replicating membrane vesicles. Here, we address the issue of the compatibility of these two replicating systems. Fatty acids form vesicles that are able to grow and divide, but vesicles composed solely of fatty acids are incompatible with the folding and activity of most ribozymes, because low concns. of divalent cations (e.g., Mg2+) cause fatty acids to ppt. Furthermore, vesicles that grow and divide must be permeable to the cations and substrates required for internal metab. We used a mixt. of myristoleic acid and its glycerol monoester to construct vesicles that were Mg2+-tolerant and found that Mg2+ cations can permeate the membrane and equilibrate within a few minutes. In vesicles encapsulating a hammerhead ribozyme, the addn. of external Mg2+ led to the activation and self-cleavage of the ribozyme mols. Vesicles composed of these amphiphiles grew spontaneously through osmotically driven competition between vesicles, and further modification of the membrane compn. allowed growth following mixed micelle addn. Our results show that membranes made from simple amphiphiles can form vesicles that are stable enough to retain encapsulated RNAs in the presence of divalent cations, yet dynamic enough to grow spontaneously and allow the passage of Mg2+ and mononucleotides without specific macromol. transporters. This combination of stability and dynamics is crit. for building model protocells in the lab. and may have been important for early cellular evolution.
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    Gorbalenya, A. E.; Enjuanes, L.; Ziebuhr, J.; Snijder, E. J. Nidovirales: Evolving the Largest RNA Virus Genome. Virus Res. 2006, 117 (1), 17– 37,  DOI: 10.1016/j.virusres.2006.01.017
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    Joyce, G. F.; Szostak, J. W. Protocells and RNA Self-Replication. Cold Spring Harb. Perspect. Biol. 2018, 10 (9), a034801,  DOI: 10.1101/cshperspect.a034801
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    Protocells and RNA self-replication
    Joyce, Gerald F.; Szostak, Jack W.
    Cold Spring Harbor Perspectives in Biology (2018), 10 (9), a034801/1-a034801/20CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
    The general notion of an "RNA world" is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA, and RNA mols. were the chief agents of catalytic function. Assuming that all of the components of RNA were available in some prebiotic locale, these components could have assembled into activated nucleotides that condensed to form RNA polymers, setting the stage for the chem. replication of polynucleotides through RNA-templated RNA polymn. If a sufficient diversity of RNAs could be copied with reasonable rate and fidelity, then Darwinian evolution would begin with RNAs that facilitated their own reprodn. enjoying a selective advantage. The concept of a "protocell" refers to a compartment where replication of the primitive genetic material took place and where primitive catalysts gave rise to products that accumulated locally for the benefit of the replicating cellular entity. Replication of both the protocell and its encapsulated genetic material would have enabled natural selection to operate based on the differential fitness of competing cellular entities, ultimately giving rise to modern cellular life.
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    Jahn, M.; Vorpahl, C.; Hübschmann, T.; Harms, H.; Müller, S. Copy Number Variability of Expression Plasmids Determined by Cell Sorting and Droplet Digital PCR. Microb. Cell Fact. 2016, 15 (1), 211,  DOI: 10.1186/s12934-016-0610-8
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    Nielsen, A. A. K.; Der, B. S.; Shin, J.; Vaidyanathan, P.; Paralanov, V.; Strychalski, E. A.; Ross, D.; Densmore, D.; Voigt, C. A. Genetic Circuit Design Automation. Science 2016, 352 (6281), aac7341,  DOI: 10.1126/science.aac7341
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    Salis, H. M.; Mirsky, E. A.; Voigt, C. A. Automated Design of Synthetic Ribosome Binding Sites to Control Protein Expression. Nat. Biotechnol. 2009, 27 (10), 946– 950,  DOI: 10.1038/nbt.1568
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    Automated design of synthetic ribosome binding sites to control protein expression
    Salis, Howard M.; Mirsky, Ethan A.; Voigt, Christopher A.
    Nature Biotechnology (2009), 27 (10), 946-950CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    Microbial engineering often requires fine control over protein expression-for example, to connect genetic circuits or control flux through a metabolic pathway. To circumvent the need for trial and error optimization, we developed a predictive method for designing synthetic ribosome binding sites, enabling a rational control over the protein expression level. Exptl. validation of >100 predictions in Escherichia coli showed that the method is accurate to within a factor of 2.3 over a range of 100,000-fold. The design method also correctly predicted that reusing identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels. We demonstrate the method's utility by rationally optimizing protein expression to connect a genetic sensor to a synthetic circuit. The proposed forward engineering approach should accelerate the construction and systematic optimization of large genetic systems.
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    Ostrov, N.; Beal, J.; Ellis, T.; Gordon, D. B.; Karas, B. J.; Lee, H. H.; Lenaghan, S. C.; Schloss, J. A.; Stracquadanio, G.; Trefzer, A.; Bader, J. S.; Church, G. M.; Coelho, C. M.; Efcavitch, J. W.; Güell, M.; Mitchell, L. A.; Nielsen, A. A. K.; Peck, B.; Smith, A. C.; Stewart, C. N., Jr; Tekotte, H. Technological Challenges and Milestones for Writing Genomes. Science 2019, 366 (6463), 310– 312,  DOI: 10.1126/science.aay0339
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    Technological challenges and milestones for writing genomes
    Ostrov, Nili; Beal, Jacob; Ellis, Tom; Gordon, D. Benjamin; Karas, Bogumil J.; Lee, Henry H.; Lenaghan, Scott C.; Schloss, Jeffery A.; Stracquadanio, Giovanni; Trefzer, Axel; Bader, Joel S.; Church, George M.; Coelho, Cintia M.; Efcavitch, J. William; Guell, Marc; Mitchell, Leslie A.; Nielsen, Alec A. K.; Peck, Bill; Smith, Alexander C.; Stewart, C. Neal, Jr.; Tekotte, Hille
    Science (Washington, DC, United States) (2019), 366 (6463), 310-312CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    A review. Engineering biol. with recombinant DNA, broadly called synthetic biol., has progressed tremendously in the last decade, owing to continued industrialization of DNA synthesis, discovery and development of mol. tools and organisms, and increasingly sophisticated modeling and analytic tools. However, we have yet to understand the full potential of engineering biol. because of our inability to write and test whole genomes, which we call synthetic genomics. Substantial improvements are needed to reduce the cost and increase the speed and reliability of genetic tools. Here, we identify emerging technologies and improvements to existing methods that will be needed in four major areas to advance synthetic genomics within the next 10 years: genome design, DNA synthesis, genome editing, and chromosome construction (see table). Similar to other large-scale projects for responsible advancement of innovative technologies, such as the Human Genome Project, an international, cross-disciplinary effort consisting of public and private entities will likely yield maximal return on investment and open new avenues of research and biotechnol.
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    Farasat, I.; Kushwaha, M.; Collens, J.; Easterbrook, M.; Guido, M.; Salis, H. M. Efficient Search, Mapping, and Optimization of Multi-Protein Genetic Systems in Diverse Bacteria. Mol. Syst. Biol. 2014, 10 (6), 731,  DOI: 10.15252/msb.20134955
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    Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria
    Farasat Iman; Easterbrook Michael; Guido Matthew; Kushwaha Manish; Collens Jason; Salis Howard M
    Molecular systems biology (2014), 10 (), 731 ISSN:.
    Developing predictive models of multi-protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi-protein expression space across a > 10,000-fold range with tailored search parameters and well-predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram-positive and gram-negative bacterial hosts. We then combined the search algorithm with system-level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence-expression-activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate-limiting steps in metabolism. Creating sequence-expression-activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs.
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    Pretorius, I. S.; Boeke, J. D. Yeast 2.0-Connecting the Dots in the Construction of the World’s First Functional Synthetic Eukaryotic Genome. FEMS Yeast Res. 2018,  DOI: 10.1093/femsyr/foy032
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    Blight, K. J.; Kolykhalov, A. A.; Rice, C. M. Efficient Initiation of HCV RNA Replication in Cell Culture. Science 2000, 290 (5498), 1972– 1974,  DOI: 10.1126/science.290.5498.1972
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    Efficient initiation of HCV RNA replication in cell culture
    Blight, Keril J.; Kolykhalov, Alexander A.; Rice, Charles M.
    Science (Washington, D. C.) (2000), 290 (5498), 1972-1974CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Hepatitis C virus (HCV) infection is a global health problem affecting an estd. 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity detg. region (ISDR). Independent of the ISDR, IFN-α rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.
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    Gibson, D. G.; Young, L.; Chuang, R.-Y.; Venter, J. C.; Hutchison, C. A., 3rd; Smith, H. O. Enzymatic Assembly of DNA Molecules up to Several Hundred Kilobases. Nat. Methods 2009, 6 (5), 343– 345,  DOI: 10.1038/nmeth.1318
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    Enzymatic assembly of DNA molecules up to several hundred kilobases
    Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan; Venter, J. Craig; Hutchison, Clyde A.; Smith, Hamilton O.
    Nature Methods (2009), 6 (5), 343-345CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    The authors describe an isothermal, single-reaction method for assembling multiple overlapping DNA mols. by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First they recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful mol. engineering tool.
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    Venter, J. C.; Glass, J. I.; Hutchison, C. A., 3rd; Vashee, S. Synthetic Chromosomes, Genomes, Viruses, and Cells. Cell 2022, 185 (15), 2708– 2724,  DOI: 10.1016/j.cell.2022.06.046
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    Synthetic chromosomes, genomes, viruses, and cells
    Venter, J. Craig; Glass, John I.; Hutchison, Clyde A. III; Vashee, Sanjay
    Cell (Cambridge, MA, United States) (2022), 185 (15), 2708-2724CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    A review. Synthetic genomics is the construction of viruses, bacteria, and eukaryotic cells with synthetic genomes. It involves two basic processes: synthesis of complete genomes or chromosomes and booting up of those synthetic nucleic acids to make viruses or living cells. The first synthetic genomics efforts resulted in the construction of viruses. This led to a revolution in viral reverse genetics and improvements in vaccine design and manuf. The first bacterium with a synthetic genome led to construction of a minimal bacterial cell and recoded Escherichia coli strains able to incorporate multiple non-std. amino acids in proteins and resistant to phage infection. Further advances led to a yeast strain with a synthetic genome and new approaches for animal and plant artificial chromosomes. On the horizon there are dramatic advances in DNA synthesis that will enable extraordinary new opportunities in medicine, industry, agriculture, and research.
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    Fredens, J.; Wang, K.; de la Torre, D.; Funke, L. F. H.; Robertson, W. E.; Christova, Y.; Chia, T.; Schmied, W. H.; Dunkelmann, D. L.; Beránek, V.; Uttamapinant, C.; Llamazares, A. G.; Elliott, T. S.; Chin, J. W. Total Synthesis of Escherichia Coli with a Recoded Genome. Nature 2019, 569 (7757), 514– 518,  DOI: 10.1038/s41586-019-1192-5
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    Total synthesis of Escherichia coli with a recoded genome
    Fredens, Julius; Wang, Kaihang; de la Torre, Daniel; Funke, Louise F. H.; Robertson, Wesley E.; Christova, Yonka; Chia, Tiongsun; Schmied, Wolfgang H.; Dunkelmann, Daniel L.; Beranek, Vaclav; Uttamapinant, Chayasith; Llamazares, Andres Gonzalez; Elliott, Thomas S.; Chin, Jason W.
    Nature (London, United Kingdom) (2019), 569 (7757), 514-518CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the no. of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme-with simple corrections at just seven positions-to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential tRNA.
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    Zhao, Y.; Coelho, C.; Hughes, A. L.; Lazar-Stefanita, L.; Yang, S.; Brooks, A. N.; Walker, R. S. K.; Zhang, W.; Lauer, S.; Hernandez, C.; Cai, J.; Mitchell, L. A.; Agmon, N.; Shen, Y.; Sall, J.; Fanfani, V.; Jalan, A.; Rivera, J.; Liang, F.-X.; Bader, J. S.; Stracquadanio, G.; Steinmetz, L. M.; Cai, Y.; Boeke, J. D. Debugging and Consolidating Multiple Synthetic Chromosomes Reveals Combinatorial Genetic Interactions. Cell 2023, 186 (24), 5220– 5236,  DOI: 10.1016/j.cell.2023.09.025
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    Woese, C. The Universal Ancestor. Proc. Natl. Acad. Sci. U. S. A. 1998, 95 (12), 6854– 6859,  DOI: 10.1073/pnas.95.12.6854
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    Woese, C. R. On the Evolution of Cells. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (13), 8742– 8747,  DOI: 10.1073/pnas.132266999
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    Sandberg, T. E.; Wise, K. S.; Dalldorf, C.; Szubin, R.; Feist, A. M.; Glass, J. I.; Palsson, B. O. Adaptive Evolution of a Minimal Organism with a Synthetic Genome. iScience 2023, 26 (9), 107500,  DOI: 10.1016/j.isci.2023.107500
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    Moger-Reischer, R. Z.; Glass, J. I.; Wise, K. S.; Sun, L.; Bittencourt, D. M. C.; Lehmkuhl, B. K.; Schoolmaster, D. R., Jr; Lynch, M.; Lennon, J. T. Evolution of a Minimal Cell. Nature 2023, 620 (7972), 122– 127,  DOI: 10.1038/s41586-023-06288-x
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    Solé, R. Revisiting Leigh Van Valen’s “A New Evolutionary Law” (1973). Biol. Theory 2022, 17 (2), 120– 125,  DOI: 10.1007/s13752-021-00391-w
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    Hammerling, M. J.; Krüger, A.; Jewett, M. C. Strategies for in Vitro Engineering of the Translation Machinery. Nucleic Acids Res. 2020, 48 (3), 1068– 1083,  DOI: 10.1093/nar/gkz1011
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    Strategies for in vitro engineering of the translation machinery
    Hammerling, Michael J.; Krueger, Antje; Jewett, Michael C.
    Nucleic Acids Research (2020), 48 (3), 1068-1083CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    A review. Engineering the process of mol. translation, or protein biosynthesis, has emerged as a major opportunity in synthetic and chem. biol. to generate novel biol. insights and enable new applications (e.g. designer protein therapeutics). Here, we review methods for engineering the process of translation in vitro. We discuss the advantages and drawbacks of the two major strategies-purified and ext.-based systems-and how they may be used to manipulate and study translation. Techniques to engineer each component of the translation machinery are covered in turn, including tRNAs, translation factors, and the ribosome. Finally, future directions and enabling technol. advances for the field are discussed.
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    Williams, T. C.; Averesch, N. J. H.; Winter, G.; Plan, M. R.; Vickers, C. E.; Nielsen, L. K.; Krömer, J. O. Quorum-Sensing Linked RNA Interference for Dynamic Metabolic Pathway Control in Saccharomyces Cerevisiae. Metab. Eng. 2015, 29, 124– 134,  DOI: 10.1016/j.ymben.2015.03.008
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    Quorum-sensing linked RNA interference for dynamic metabolic pathway control in Saccharomyces cerevisiae
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    Metabolic Engineering (2015), 29 (), 124-134CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)
    Some of the most productive metabolic engineering strategies involve genetic modifications that cause severe metabolic burden on the host cell. Growth-limiting genetic modifications can be more effective if they are 'switched on' after a population growth phase has been completed. To address this problem we have engineered dynamic regulation using a previously developed synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit autonomously triggers gene expression at a high population d., and was linked with an RNA interference module to enable target gene silencing. As a demonstration the circuit was used to control flux through the shikimate pathway for the prodn. of para-hydroxybenzoic acid (PHBA). Dynamic RNA repression allowed gene knock-downs which were identified by elementary flux mode anal. as highly productive but with low biomass formation to be implemented after a population growth phase, resulting in the highest published PHBA titer in yeast (1.1 mM).
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    Robinson, A. O.; Venero, O. M.; Adamala, K. P. Toward Synthetic Life: Biomimetic Synthetic Cell Communication. Curr. Opin. Chem. Biol. 2021, 64, 165– 173,  DOI: 10.1016/j.cbpa.2021.08.008
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    Toward synthetic life: Biomimetic synthetic cell communication
    Robinson, Abbey O.; Venero, Orion M.; Adamala, Katarzyna P.
    Current Opinion in Chemical Biology (2021), 64 (), 165-173CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    A review. Engineering synthetic minimal cells provide a controllable chassis for studying the biochem. principles of natural life, increasing our understanding of complex biol. processes. Recently, synthetic cell engineering has enabled communication between both natural live cells and other synthetic cells. A system such as these enable studying interactions between populations of cells, both natural and artificial, and engineering small mol. cell communication protocols for a variety of basic research and practical applications. In this review, we summarize recent progress in engineering communication between synthetic and natural cells, and we speculate about the possible future directions of this work.
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    Garamella, J.; Majumder, S.; Liu, A. P.; Noireaux, V. An Adaptive Synthetic Cell Based on Mechanosensing, Biosensing, and Inducible Gene Circuits. ACS Synth. Biol. 2019, 8 (8), 1913– 1920,  DOI: 10.1021/acssynbio.9b00204
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    An Adaptive Synthetic Cell Based on Mechanosensing, Biosensing, and Inducible Gene Circuits
    Garamella, Jonathan; Majumder, Sagardip; Liu, Allen P.; Noireaux, Vincent
    ACS Synthetic Biology (2019), 8 (8), 1913-1920CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    The bottom-up assembly of synthetic cell systems capable of recapitulating biol. functions has become a means to understand living matter by construction. The integration of biomol. components into active, cell-sized, genetically programmed compartments remains, however, a major bottleneck for building synthetic cells. A primary feature of real cells is their ability to actively interact with their surroundings, particularly in stressed conditions. Here, we construct a synthetic cell equipped with an inducible genetic circuit that responds to changes in osmotic pressure through the mechanosensitive channel MscL. Liposomes loaded with an E. coli cell-free transcription-translation (TXTL) system are induced with IPTG when exposed to hypo-osmotic soln., resulting in the expression of a bacterial cytoskeletal protein MreB. MreB assocs. with the membrane to generate a cortex-like structure. Our work provides the first example of mol. integration that couples mechanosensitivity, gene expression, and self-assembly at the inner membrane of synthetic cells.
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    Liu, H.; Yang, Q.; Peng, R.; Kuai, H.; Lyu, Y.; Pan, X.; Liu, Q.; Tan, W. Artificial Signal Feedback Network Mimicking Cellular Adaptivity. J. Am. Chem. Soc. 2019, 141 (16), 6458– 6461,  DOI: 10.1021/jacs.8b13816
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    Artificial Signal Feedback Network Mimicking Cellular Adaptivity
    Liu, Hui; Yang, Qiuxia; Peng, Ruizi; Kuai, Hailan; Lyu, Yifan; Pan, Xiaoshu; Liu, Qiaoling; Tan, Weihong
    Journal of the American Chemical Society (2019), 141 (16), 6458-6461CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Inspired by this elegant system of cellular adaptivity, we herein report the rational design of a dynamic artificial adaptive system able to sense and respond to environmental stresses in a unique sense-and-respond mode. Utilizing DNA nanotechnol., we constructed an artificial signal feedback network and anchored it to surface membrane of a model giant membrane vesicle (GMV) protocell. Such system would need to both sense incoming stimuli and emit a feedback response to eliminate the stimuli. To accomplish this mechanistically, our DNA-based artificial signal system, hereinafter termed DASsys, was equipped with a DNA trig-ger-induced DNA polymer formation and dissocn. machinery. Thus, through a sequential cascade of stimulus-induced DNA strand displacement, DASsys could effectively sense and respond to incoming stimuli. Then, by eliminating the stimulus, the membrane surface would return to its initial state, realizing the formation of a cyclical feedback mechanism. Overall, our strategy opens up a route to the construction of artificial signaling system capable of maintaining homeostasis in the cellular micromilieu, and addresses important emerging challenges in bioinspired engineering.
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    Gispert, I.; Hindley, J. W.; Pilkington, C. P.; Shree, H.; Barter, L. M. C.; Ces, O.; Elani, Y. Stimuli-Responsive Vesicles as Distributed Artificial Organelles for Bacterial Activation. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 (42), e2206563119,  DOI: 10.1073/pnas.2206563119
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    Aufinger, L.; Simmel, F. C. Establishing Communication Between Artificial Cells. Chemistry 2019, 25 (55), 12659– 12670,  DOI: 10.1002/chem.201901726
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    Establishing Communication Between Artificial Cells
    Aufinger, Lukas; Simmel, Friedrich C.
    Chemistry - A European Journal (2019), 25 (55), 12659-12670CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Communication between artificial cells is essential for the realization of complex dynamical behaviors at the multi-cell level. It is also an important prerequisite for modular systems design, because it dets. how spatially sepd. functional modules can coordinate their actions. Among others, mol. communication is required for artificial cell signaling, synchronization of cellular behaviors, computation, group-level decision-making processes and pattern formation in artificial tissues. In this review, an overview of various recent approaches to create communicating artificial cellular systems is provided. In this context, important physicochem. boundary conditions that have to be considered for the design of the communicating cells are also described, and a survey of the most striking emergent behaviors that may be achieved in such systems is given.
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    Pereyre, S.; Sirand-Pugnet, P.; Beven, L.; Charron, A.; Renaudin, H.; Barré, A.; Avenaud, P.; Jacob, D.; Couloux, A.; Barbe, V.; de Daruvar, A.; Blanchard, A.; Bébéar, C. Life on Arginine for Mycoplasma Hominis: Clues from Its Minimal Genome and Comparison with Other Human Urogenital Mycoplasmas. PLoS Genet. 2009, 5 (10), e1000677,  DOI: 10.1371/journal.pgen.1000677
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    Li, S.; Guan, J.-L.; Chien, S. Biochemistry and Biomechanics of Cell Motility. Annu. Rev. Biomed. Eng. 2005, 7, 105– 150,  DOI: 10.1146/annurev.bioeng.7.060804.100340
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    Biochemistry and biomechanics of cell motility
    Li, Song; Guan, Jun-Lin; Chien, Shu
    Annual Review of Biomedical Engineering (2005), 7 (), 105-150, 2 platesCODEN: ARBEF7; ISSN:1523-9829. (Annual Reviews Inc.)
    A review. Cell motility is an essential cellular process for a variety of biol. events. The process of cell migration requires the integration and coordination of complex biochem. and biomech. signals. The protrusion force at the leading edge of a cell is generated by the cytoskeleton, and this force generation is controlled by multiple signaling cascades. The formation of new adhesions at the front and the release of adhesions at the rear involve the outside-in and inside-out signaling mediated by integrins and other adhesion receptors. The traction force generated by the cell on the extracellular matrix (ECM) regulates cell-ECM adhesions, and the counter force exerted by ECM on the cell drives the migration. The polarity of cell migration can be amplified and maintained by the feedback loop between the cytoskeleton and cell-ECM adhesions. Cell migration in 3-dimensional ECM has characteristics distinct from that on 2-dimensional ECM. The migration of cells is initiated and modulated by external chem. and mech. factors, such as chemoattractants and the mech. forces acting on the cells and ECM, as well as the surface d., distribution, topog., and rigidity of the ECM.
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    Brunet, T.; Albert, M.; Roman, W.; Coyle, M. C.; Spitzer, D. C.; King, N. A Flagellate-to-Amoeboid Switch in the Closest Living Relatives of Animals. eLife 2021,  DOI: 10.7554/eLife.61037
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    Siton-Mendelson, O.; Bernheim-Groswasser, A. Toward the Reconstitution of Synthetic Cell Motility. Cell Adh. Migr. 2016, 10 (5), 461– 474,  DOI: 10.1080/19336918.2016.1170260
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    Toward the reconstitution of synthetic cell motility
    Siton-Mendelson Orit; Bernheim-Groswasser Anne
    Cell adhesion & migration (2016), 10 (5), 461-474 ISSN:.
    Cellular motility is a fundamental process essential for embryonic development, wound healing, immune responses, and tissues development. Cells are mostly moving by crawling on external, or inside, substrates which can differ in their surface composition, geometry, and dimensionality. Cells can adopt different migration phenotypes, e.g., bleb-based and protrusion-based, depending on myosin contractility, surface adhesion, and cell confinement. In the few past decades, research on cell motility has focused on uncovering the major molecular players and their order of events. Despite major progresses, our ability to infer on the collective behavior from the molecular properties remains a major challenge, especially because cell migration integrates numerous chemical and mechanical processes that are coupled via feedbacks that span over large range of time and length scales. For this reason, reconstituted model systems were developed. These systems allow for full control of the molecular constituents and various system parameters, thereby providing insight into their individual roles and functions. In this review we describe the various reconstituted model systems that were developed in the past decades. Because of the multiple steps involved in cell motility and the complexity of the overall process, most of the model systems focus on very specific aspects of the individual steps of cell motility. Here we describe the main advancement in cell motility reconstitution and discuss the main challenges toward the realization of a synthetic motile cell.
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    Blanken, D.; van Nies, P.; Danelon, C. Quantitative Imaging of Gene-Expressing Liposomes Reveals Rare Favorable Phenotypes. Phys. Biol. 2019, 16 (4), 045002,  DOI: 10.1088/1478-3975/ab0c62
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    Quantitative imaging of gene-expressing liposomes reveals rare favorable phenotypes
    Blanken, Duco; van Nies, Pauline; Danelon, Christophe
    Physical Biology (2019), 16 (4), 045002CODEN: PBHIAT; ISSN:1478-3975. (IOP Publishing Ltd.)
    We report on direct imaging of tens of thousands of gene-expressing liposomes per sample allowing us to assess sub-population features in a statistically relevant manner. Both the vesicle size(diam. <10μm) and lipid compn. (mixt. of phospholipids with zwitterionic and neg. charged headgroups, including cardiolipin) are compatible with the properties of bacterial cells. Therefore, our liposomes provide suitable chassis to host Escherichia coli-derived PURE translation machinery and other bacterial processes in future developments. The potential of high-content imaging to identify rare phenotypes is demonstrated by fact that a subset of the liposome population exhibits remarkably high yield of synthesized protein or prolonged expression lifespan that surpasses performance of ensemble liposome-averaged and bulk reactions. Among the three com. PURE systems tested, PUREfrex2.0 offers the most favorable phenotypes displaying both high yield and long protein synthesis lifespan. Moreover, probing membrane permeability reveals a large heterogeneity amongst liposomes. In situ expression and membrane embedding of the pore-forming connexin leads to a characteristic permeability time profile, while increasing the fraction of permeable liposomes in the population. We see diversity in gene expression dynamics and membrane permeability as an opportunity to complement a rational design approach aiming at further implementing biol. functions in liposome-based synthetic cells.
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    Stano, P.; D’Aguanno, E.; Bolz, J.; Fahr, A.; Luisi, P. L. A Remarkable Self-Organization Process as the Origin of Primitive Functional Cells. Angew. Chem., Int. Ed. Engl. 2013, 52 (50), 13397– 13400,  DOI: 10.1002/anie.201306613
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    Weitz, M.; Kim, J.; Kapsner, K.; Winfree, E.; Franco, E.; Simmel, F. C. Diversity in the Dynamical Behaviour of a Compartmentalized Programmable Biochemical Oscillator. Nat. Chem. 2014, 6 (4), 295– 302,  DOI: 10.1038/nchem.1869
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    Diversity in the dynamical behaviour of a compartmentalized programmable biochemical oscillator
    Weitz, Maximilian; Kim, Jongmin; Kapsner, Korbinian; Winfree, Erik; Franco, Elisa; Simmel, Friedrich C.
    Nature Chemistry (2014), 6 (4), 295-302CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    In vitro compartmentalization of biochem. reaction networks is a crucial step towards engineering artificial cell-scale devices and systems. At this scale the dynamics of mol. systems becomes stochastic, which introduces several engineering challenges and opportunities. Here we study a programmable transcriptional oscillator system that is compartmentalized into microemulsion droplets with vols. between 33 fl and 16 pl. Simultaneous measurement of large populations of droplets reveals major variations in the amplitude, frequency and damping of the oscillations. Variability increases for smaller droplets and depends on the operating point of the oscillator. Rather than reflecting the stochastic kinetics of the chem. reaction network itself, the variability can be attributed to the statistical variation of reactant concns. created during their partitioning into droplets. We anticipate that robustness to partitioning variability will be a crit. challenge for engineering cell-scale systems, and that highly parallel time-series acquisition from microemulsion droplets will become a key tool for characterization of stochastic circuit function.
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    Hansen, M. M. K.; Meijer, L. H. H.; Spruijt, E.; Maas, R. J. M.; Rosquelles, M. V.; Groen, J.; Heus, H. A.; Huck, W. T. S. Macromolecular Crowding Creates Heterogeneous Environments of Gene Expression in Picolitre Droplets. Nat. Nanotechnol. 2016, 11 (2), 191– 197,  DOI: 10.1038/nnano.2015.243
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    Macromolecular crowding creates heterogeneous environments of gene expression in picolitre droplets
    Hansen, Maike M. K.; Meijer, Lenny H. H.; Spruijt, Evan; Maas, Roel J. M.; Rosquelles, Marta Ventosa; Groen, Joost; Heus, Hans A.; Huck, Wilhelm T. S.
    Nature Nanotechnology (2016), 11 (2), 191-197CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    Understanding the dynamics of complex enzymic reactions in highly crowded small vols. is crucial for the development of synthetic minimal cells. Compartmentalized biochem. reactions in cell-sized containers exhibit a degree of randomness due to the small no. of mols. involved. However, it is unknown how the phys. environment contributes to the stochastic nature of multistep enzymic processes. Here, we present a robust method to quantify gene expression noise in vitro using droplet microfluidics. We study the changes in stochasticity in the cell-free gene expression of two genes compartmentalized within droplets as a function of DNA copy no. and macromol. crowding. We find that decreased diffusion caused by a crowded environment leads to the spontaneous formation of heterogeneous microenvironments of mRNA as local prodn. rates exceed the diffusion rates of macromols. This heterogeneity leads to a higher probability of the mol. machinery staying in the same microenvironment, directly increasing the system's stochasticity.
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    Rao, C. V. Expanding the Synthetic Biology Toolbox: Engineering Orthogonal Regulators of Gene Expression. Curr. Opin. Biotechnol. 2012, 23 (5), 689– 694,  DOI: 10.1016/j.copbio.2011.12.015
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    Yelleswarapu, M.; van der Linden, A. J.; van Sluijs, B.; Pieters, P. A.; Dubuc, E.; de Greef, T. F. A.; Huck, W. T. S. Sigma Factor-Mediated Tuning of Bacterial Cell-Free Synthetic Genetic Oscillators. ACS Synth. Biol. 2018, 7 (12), 2879– 2887,  DOI: 10.1021/acssynbio.8b00300
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    Sigma factor-mediated tuning of bacterial cell-free synthetic genetic oscillators
    Yelleswarapu, Maaruthy; van der Linden, Ardjan J.; van Sluijs, Bob; Pieters, Pascal A.; Dubuc, Emilien; de Greef, Tom F. A.; Huck, Wilhelm T. S.
    ACS Synthetic Biology (2018), 7 (12), 2879-2887CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Cell-free transcription-translation provides a simplified prototyping environment to rapidly design and study synthetic networks. Despite the presence of a well-characterized toolbox of genetic elements, examples of genetic networks that exhibit complex temporal behavior are scarce. Here, we present a genetic oscillator implemented in an Escherichia coli-based cell-free system under steady-state conditions using microfluidic flow reactors. The oscillator had an activator-repressor motif that utilizes the native transcriptional machinery of E. coli: the RNA polymerase (RNAP) and its assocd. σ-factors. We optimized a kinetic model with exptl. data using an evolutionary algorithm to quantify the key regulatory model parameters. The functional modulation of RNAP was investigated by coupling 2 oscillators driven by competing σ-factors, allowing the modification of network properties by means of passive transcriptional regulation.
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    Li, J.; Zhang, C.; Huang, P.; Kuru, E.; Forster-Benson, E. T. C.; Li, T.; Church, G. M. Dissecting Limiting Factors of the Protein Synthesis Using Recombinant Elements (PURE) System. Translation (Austin) 2017, 5 (1), e1327006,  DOI: 10.1080/21690731.2017.1327006
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    Lavickova, B.; Maerkl, S. J. A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System. ACS Synth. Biol. 2019, 8 (2), 455– 462,  DOI: 10.1021/acssynbio.8b00427
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    A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System
    Lavickova, Barbora; Maerkl, Sebastian J.
    ACS Synthetic Biology (2019), 8 (2), 455-462CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription-translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.
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    Li, J.; Gu, L.; Aach, J.; Church, G. M. Improved Cell-Free RNA and Protein Synthesis System. PLoS One 2014, 9 (9), e106232,  DOI: 10.1371/journal.pone.0106232
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    Improved cell-free RNA and protein synthesis system
    Li, Jun; Gu, Liangcai; Aach, John; Church, George M.
    PLoS One (2014), 9 (9), e106232/1-e106232/11, 11 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Cell-free RNA and protein synthesis (CFPS) is becoming increasingly used for protein prodn. as yields increase and costs decrease. Advances in reconstituted CFPS systems such as the Protein synthesis Using Recombinant Elements (PURE) system offer new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, protein microarrays, isotopic labeling, and incorporating unnatural amino acids. In this study, using firefly luciferase synthesis as a reporter system, we improved PURE system productivity up to 5 fold by adding or adjusting a variety of factors that affect transcription and translation, including Elongation factors (EF-Ts, EF-Tu, EF-G and EF4), ribosome recycling factor (RRF), release factors (RF1, RF2, RF3), chaperones (GroEL/ES), BSA and tRNAs. The work provides a more efficient defined in vitro transcription and translation system and a deeper understanding of the factors that limit the whole system efficiency.
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    Jewett, M. C.; Calhoun, K. A.; Voloshin, A.; Wuu, J. J.; Swartz, J. R. An Integrated Cell-Free Metabolic Platform for Protein Production and Synthetic Biology. Mol. Syst. Biol. 2008, 4, 220,  DOI: 10.1038/msb.2008.57
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    An integrated cell-free metabolic platform for protein production and synthetic biology
    Jewett Michael C; Calhoun Kara A; Voloshin Alexei; Wuu Jessica J; Swartz James R
    Molecular systems biology (2008), 4 (), 220 ISSN:.
    Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements.
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  181. 181
    Silverman, A. D.; Karim, A. S.; Jewett, M. C. Cell-Free Gene Expression: An Expanded Repertoire of Applications. Nat. Rev. Genet. 2020, 21 (3), 151– 170,  DOI: 10.1038/s41576-019-0186-3
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    Cell-free gene expression: an expanded repertoire of applications
    Silverman, Adam D.; Karim, Ashty S.; Jewett, Michael C.
    Nature Reviews Genetics (2020), 21 (3), 151-170CODEN: NRGAAM; ISSN:1471-0056. (Nature Research)
    Cell-free biol. is the activation of biol. processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent tech. renaissance has facilitated high-yielding (grams of protein per L), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biol. design. These advances provide exciting opportunities to profoundly transform synthetic biol. by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compds., on-demand biomanufg., building cells from the bottom up, and next-generation educational kits.
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  182. 182
    Ouyang, X.; Zhou, X.; Lai, S. N.; Liu, Q.; Zheng, B. Immobilization of Proteins of Cell Extract to Hydrogel Networks Enhances the Longevity of Cell-Free Protein Synthesis and Supports Gene Networks. ACS Synth. Biol. 2021, 10 (4), 749– 755,  DOI: 10.1021/acssynbio.0c00541
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    New England Biolabs. NEBExpress Cell-free ecoli Protein Synthesis System. https://www.neb.com/products/e5360-nebexpress-cell-free-ecoli-protein-synthesis-system (accessed on August 21, 2023).
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    Zimmerman, S. B.; Trach, S. O. Estimation of Macromolecule Concentrations and Excluded Volume Effects for the Cytoplasm of Escherichia Coli. J. Mol. Biol. 1991, 222 (3), 599– 620,  DOI: 10.1016/0022-2836(91)90499-V
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    184
    Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli
    Zimmerman, Steven B.; Trach, Stefan O.
    Journal of Molecular Biology (1991), 222 (3), 599-620CODEN: JMOBAK; ISSN:0022-2836.
    The very high concn. of macromols. within cells can potentially have an overwhelming effect on the thermodn. activity of cellular components because of excluded vol. effects. To est. the magnitudes of such effects, an exptl. study was made of the cytoplasm of Escherichia coli. Parameters from cells and cell exts. are used to calc. approx. activity coeffs. for cytoplasmic conditions. These calcns. require a representation of the sizes, concns. and effective sp. vol. of the macromols. in the exts. Macromol. size representations are obtained either by applying a two-phase distribution assay to define a related homogeneous soln. or by using the mol. mass distribution of macromols. from gel filtration. Macromol. concns. in cytoplasm are obtained from analyses of exts. by applying a correction for the diln. that occurs during extn. That factor is detd. from expts. based upon the known impermeability of the cytoplasmic vol. to sucrose in intact E. coli. Macromol. concns. in the cytoplasm of E. coli in either exponential or stationary growth phase are estd. to be ≈0.3 to 0.4 g/mL. Macromol. sp. vol. are inferred from the compn. of close-packed ppts. induced by polyethylene glycol. Several well-characterized proteins which bind to DNA (lac repressor, RNA polymerase) are extremely sensitive to changes in salt concn. in studies in vitro, but are insensitive in studies in vivo. Application of the activity coeffs. from the present work indicates that at least part of this discrepancy arises from the differences in excluded vols. in these studies. Applications of the activity coeffs. to soly. or to assocn. reactions are also discussed, as are changes assocd. with cell growth phase and osmotic or other effects. The use of solns. of purified macromols. that emulate the crowding conditions inferred for cytoplasm is discussed.
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  185. 185
    Record, M. T., Jr; Courtenay, E. S.; Cayley, D. S.; Guttman, H. J. Responses of E. Coli to Osmotic Stress: Large Changes in Amounts of Cytoplasmic Solutes and Water. Trends Biochem. Sci. 1998, 23 (4), 143– 148,  DOI: 10.1016/S0968-0004(98)01196-7
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    185
    Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water
    Record, M. Thomas, Jr.; Courtenay, Elizabeth S.; Cayley, D. Scott; Guttman, Harry J.
    Trends in Biochemical Sciences (1998), 23 (4), 143-148CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Science Ltd.)
    A review with 37 refs. Escherichia coli is capable of growing in environments ranging from very dil. aq. solns. of essential nutrients to media contg. molar concns. of salts or nonelectrolyte solutes. Growth in environments with such a wide range (at least 100-fold) of osmolarities poses significant physiol. challenges for cells. To meet these challenges, E. coli adjusts a wide range of cytoplasmic soln. variables, including the cytoplasmic amts. both of water and of charged and uncharged solutes.
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  186. 186
    Cayley, S.; Lewis, B. A.; Guttman, H. J.; Record, M. T. Jr. Characterization of the Cytoplasm of Escherichia Coli K-12 as a Function of External Osmolarity. Implications for Protein-DNA Interactions in Vivo. J. Mol. Biol. 1991, 222 (2), 281– 300,  DOI: 10.1016/0022-2836(91)90212-O
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    Li, J.; Haas, W.; Jackson, K.; Kuru, E.; Jewett, M. C.; Fan, Z. H.; Gygi, S.; Church, G. M. Cogenerating Synthetic Parts toward a Self-Replicating System. ACS Synth. Biol. 2017, 6 (7), 1327– 1336,  DOI: 10.1021/acssynbio.6b00342
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    187
    Co-generating Synthetic Parts toward a Self-Replicating System
    Li, Jun; Haas, Wilhelm; Jackson, Kirsten; Kuru, Erkin; Jewett, Michael C.; Fan, Z. Hugh; Gygi, Steven; Church, George M.
    ACS Synthetic Biology (2017), 6 (7), 1327-1336CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    To build replicating systems with new functions, the engineering of existing biol. machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE might be the basis for a radically alterable, lifelike system after optimization. Here, the authors regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. The authors show that using stable isotope labeling with amino acids, mass spectrometry based quant. proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when coexpressed in batch format PURE system. By optimizing DNA template concns. and adapting a miniaturized Fluid Array Device with optimized feeding soln., the authors were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in coexpression pool varied from ∼1.5 to 5-fold compared to the batch mode, with up to ∼2.4 μM yield for a single r-protein. Reconstituted ribosomes under physiol. condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ∼13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. This work also points to what is still needed to obtain self-replicating synthetic ribosomes in situ in the PURE system.
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  188. 188
    Murase, Y.; Nakanishi, H.; Tsuji, G.; Sunami, T.; Ichihashi, N. In Vitro Evolution of Unmodified 16S rRNA for Simple Ribosome Reconstitution. ACS Synth. Biol. 2018, 7 (2), 576– 583,  DOI: 10.1021/acssynbio.7b00333
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    Jewett, M. C.; Fritz, B. R.; Timmerman, L. E.; Church, G. M. In Vitro Integration of Ribosomal RNA Synthesis, Ribosome Assembly, and Translation. Mol. Syst. Biol. 2013, 9, 678,  DOI: 10.1038/msb.2013.31
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    189
    In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation
    Jewett, Michael C.; Fritz, Brian R.; Timmerman, Laura E.; Church, George M.
    Molecular Systems Biology (2013), 9 (), 678CODEN: MSBOC3; ISSN:1744-4292. (Nature Publishing Group)
    Purely in vitro ribosome synthesis could provide a crit. step towards unraveling the systems biol. of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli exts. While conventional methods for E. coli ribosome reconstitution are non-physiol., our approach attempts to mimic chem. conditions in the cytoplasm, thus permitting several biol. processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technol. enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties.
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  190. 190
    Fritz, B. R.; Jamil, O. K.; Jewett, M. C. Implications of Macromolecular Crowding and Reducing Conditions for in Vitro Ribosome Construction. Nucleic Acids Res. 2015, 43 (9), 4774– 4784,  DOI: 10.1093/nar/gkv329
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    190
    Implications of macromolecular crowding and reducing conditions for in vitro ribosome construction
    Fritz, Brian R.; Jamil, Osman K.; Jewett, Michael C.
    Nucleic Acids Research (2015), 43 (9), 4774-4784CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    The in vitro construction of Escherichia coli ribosomes could elucidate a deeper understanding of these complex mol. machines and make possible the prodn. of synthetic variants with new functions. Toward this goal, the authors recently developed an integrated synthesis, assembly, and translation (iSAT) system that allows for co-activation of rRNA transcription and ribosome assembly, mRNA transcription, and protein translation without intact cells. Here, the authors discovered that macromol. crowding and reducing agents increased overall iSAT protein synthesis; the combination of 6% Ficoll 400 and 2 mM DTBA yielded an approx. 5-fold increase in overall iSAT protein synthesis activity. By utilizing a fluorescent RNA aptamer, fluorescent reporter proteins, and ribosome sedimentation anal., the authors showed that crowding agents increased iSAT yields by enhancing translation while reducing agents increased rRNA transcription and ribosome assembly. Finally, the authors showed that iSAT ribosomes possessed ∼70% of the protein synthesis activity of in vivo-assembled E. coli ribosomes. This work improved iSAT protein synthesis through the addn. of crowding and reducing agents, provides a thorough understanding of the effect of these additives within the iSAT system and demonstrated how iSAT allows for manipulation and anal. of ribosome biogenesis in the context of an in vitro transcription-translation system.
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  191. 191
    Hammerling, M. J.; Fritz, B. R.; Yoesep, D. J.; Kim, D. S.; Carlson, E. D.; Jewett, M. C. In Vitro Ribosome Synthesis and Evolution through Ribosome Display. Nat. Commun. 2020, 11 (1), 1108,  DOI: 10.1038/s41467-020-14705-2
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    191
    In-vitro ribosome synthesis and evolution through ribosome display
    Hammerling, Michael J.; Fritz, Brian R.; Yoesep, Danielle J.; Kim, Do Soon; Carlson, Erik D.; Jewett, Michael C.
    Nature Communications (2020), 11 (1), 1108CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in-vitro methodol. for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ∼1.7 X 107 member library of rRNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ∼4 X 103 rRNA variants. We further demonstrate the prevalence of pos. epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of mol. translation and enable selection of ribosomes with altered properties.
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  192. 192
    Wang, T.; Lu, Y. Toward Minimal Transcription-Translation Machinery. ACS Synth. Biol. 2023, 12 (11), 3312– 3327,  DOI: 10.1021/acssynbio.3c00324
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    Oberholzer, T.; Albrizio, M.; Luisi, P. L. Polymerase Chain Reaction in Liposomes. Chem. Biol. 1995, 2 (10), 677– 682,  DOI: 10.1016/1074-5521(95)90031-4
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    Polymerase chain reaction in liposomes
    Oberholzer, Thomas; Albrizio, Maria; Luisi, Pier Luigi
    Chemistry & Biology (1995), 2 (10), 677-82CODEN: CBOLE2; ISSN:1074-5521. (Current Biology)
    Background: Compartmentalization of biochem. reactions within a spherically closed bilayer is an important step in the mol. evolution of cells. Liposomes are the most suitable structures to model this kind of chem. We have used the polymerase chain reaction (PCR) to demonstrate that complex biochem. reactions such as DNA replication can be carried out inside these compartments. Results: We describe the first example of DNA amplification by the PCR occurring inside liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or of a mixt. of POPC and phosphatidylserine. We show that these liposomes are stable even under the high temp. conditions used for PCR. Although only a very small fraction of liposomes contains all eight different reagents together, a significant amt. of DNA is produced which can be obsd. by polyacrylamide gel electrophoresis. Conclusions: This work shows that it is possible to carry out complex biochem. reactions within liposomes, which may be germane to the question of the origin of living cells. We have established the parameters and conditions that are crit. for carrying out this complex reaction within the liposome compartment.
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  194. 194
    Kawska, A.; Carvalho, K.; Manzi, J.; Boujemaa-Paterski, R.; Blanchoin, L.; Martiel, J.-L.; Sykes, C. How Actin Network Dynamics Control the Onset of Actin-Based Motility. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (36), 14440– 14445,  DOI: 10.1073/pnas.1117096109
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    Hürtgen, D.; Härtel, T.; Murray, S. M.; Sourjik, V.; Schwille, P. Functional Modules of Minimal Cell Division for Synthetic Biology. Adv. Biosyst. 2019, 3 (6), e1800315,  DOI: 10.1002/adbi.201800315
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    Pelletier, J. F.; Sun, L.; Wise, K. S.; Assad-Garcia, N.; Karas, B. J.; Deerinck, T. J.; Ellisman, M. H.; Mershin, A.; Gershenfeld, N.; Chuang, R.-Y.; Glass, J. I.; Strychalski, E. A. Genetic Requirements for Cell Division in a Genomically Minimal Cell. Cell 2021, 184 (9), 2430– 2440,  DOI: 10.1016/j.cell.2021.03.008
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    196
    Genetic requirements for cell division in a genomically minimal cell
    Pelletier, James F.; Sun, Lijie; Wise, Kim S.; Assad-Garcia, Nacyra; Karas, Bogumil J.; Deerinck, Thomas J.; Ellisman, Mark H.; Mershin, Andreas; Gershenfeld, Neil; Chuang, Ray-Yuan; Glass, John I.; Strychalski, Elizabeth A.
    Cell (Cambridge, MA, United States) (2021), 184 (9), 2430-2440.e16CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiol. processes. Although this minimal cell includes genes essential for population growth, the physiol. of its single cells remained uncharacterized. To investigate striking morphol. variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and det. genes affecting cell morphol. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphol. similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-assocd. proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphol. in a genomically minimal cell. The fully annotated genome sequence of JCVI-syn3A is deposited in NCBI (GenBank: CP016816.2).
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    Zhang, S.; Contini, C.; Hindley, J. W.; Bolognesi, G.; Elani, Y.; Ces, O. Engineering Motile Aqueous Phase-Separated Droplets via Liposome Stabilisation. Nat. Commun. 2021, 12 (1), 1673,  DOI: 10.1038/s41467-021-21832-x
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    197
    Engineering motile aqueous phase-separated droplets via liposome stabilisation
    Zhang, Shaobin; Contini, Claudia; Hindley, James W.; Bolognesi, Guido; Elani, Yuval; Ces, Oscar
    Nature Communications (2021), 12 (1), 1673CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    There are increasing efforts to engineer functional compartments that mimic cellular behaviors from the bottom-up. One behavior that is receiving particular attention is motility, due to its biotechnol. potential and ubiquity in living systems. Many existing platforms make use of the Marangoni effect to achieve motion in water/oil (w/o) droplet systems. However, most of these systems are unsuitable for biol. applications due to biocompatibility issues caused by the presence of oil phases. Here we report a biocompatible all aq. (wt./wt.) PEG/dextran Pickering-like emulsion system consisting of liposome-stabilized cell-sized droplets, where the stability can be easily tuned by adjusting liposome compn. and concn. We demonstrate that the compartments are capable of neg. chemotaxis: these droplets can respond to a PEG/dextran polymer gradient through directional motion down to the gradient. The biocompatibility, motility and partitioning abilities of this droplet system offers new directions to pursue research in motion-related biol. processes.
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  198. 198
    Li, M.; Brinkmann, M.; Pagonabarraga, I.; Seemann, R.; Fleury, J.-B. Spatiotemporal Control of Cargo Delivery Performed by Programmable Self-Propelled Janus Droplets. Commun. Phys. 2018, 1 (1), 1– 8,  DOI: 10.1038/s42005-018-0025-4
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    198
    Breakdown of diffusivity-entropy scaling in colloidal glass-forming liquids
    Li, Bo; Xiao, Xiuming; Lou, Kai; Wang, Shuxia; Wen, Weijia; Wang, Ziren
    Communications Physics (2018), 1 (1), 1-9CODEN: CPOHDJ; ISSN:2399-3650. (Nature Research)
    Glass is a liq. that has lost its ability to flow. Why this particular substance undergoes such a dramatic kinetic slowdown yet remains barely distinguishable in structure from its fluid state upon cooling constitutes the central question of glass transition physics. Here, we investigate the pathway of kinetic slowdown in glass-forming liqs. that consist of monolayers of ellipsoidal or binary spherical colloids. In contrast to rotational motion, the dynamics of the translational motion begin to violently slow down at considerably low area fractions (.vphi.T). At .vphi.T, anomalous translation-rotation coupling is enhanced and the topog. of the free energy landscape become rugged. Based on the pos. correlation between .vphi.T and fragility, the measurement of .vphi.T offers a novel method for predicting glassy dynamics, circumventing the prohibitive increase in equil. times required in high-d. regions. Our results highlight the role that thermodynamical entropy plays in glass transitions.
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  199. 199
    Litschel, T.; Kelley, C. F.; Holz, D.; Adeli Koudehi, M.; Vogel, S. K.; Burbaum, L.; Mizuno, N.; Vavylonis, D.; Schwille, P. Reconstitution of Contractile Actomyosin Rings in Vesicles. Nat. Commun. 2021, 12 (1), 2254,  DOI: 10.1038/s41467-021-22422-7
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    199
    Reconstitution of contractile actomyosin rings in vesicles
    Litschel, Thomas; Kelley, Charlotte F.; Holz, Danielle; Adeli Koudehi, Maral; Vogel, Sven K.; Burbaum, Laura; Mizuno, Naoko; Vavylonis, Dimitrios; Schwille, Petra
    Nature Communications (2021), 12 (1), 2254CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    One of the grand challenges of bottom-up synthetic biol. is the development of minimal machineries for cell division. The mech. transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the mol. scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theor. modeling. By changing few key parameters, actin polymn. can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theor. considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.
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  200. 200
    Bashirzadeh, Y.; Moghimianavval, H.; Liu, A. P. Encapsulated Actomyosin Patterns Drive Cell-like Membrane Shape Changes. iScience 2022, 25 (5), 104236,  DOI: 10.1016/j.isci.2022.104236
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    200
    Encapsulated actomyosin patterns drive cell-like membrane shape changes
    Bashirzadeh, Yashar; Moghimianavval, Hossein; Liu, Allen P.
    iScience (2022), 25 (5), 104236CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)
    Cell shape changes from locomotion to cytokinesis are, to a large extent, driven by myosin-driven remodeling of cortical actin patterns. Passive crosslinkers such as α-actinin and fascin as well as actin nucleator Arp2/3 complex largely det. actin network architecture and, consequently, membrane shape changes. Here we reconstitute actomyosin networks inside cell-sized lipid bilayer vesicles and show that depending on vesicle size and concns. of α-actinin and fascin actomyosin networks assemble into ring and aster-like patterns. Anchoring actin to the membrane does not change actin network architecture yet exerts forces and deforms the membrane when assembled in the form of a contractile ring. In the presence of α-actinin and fascin, an Arp2/3 complex-mediated actomyosin cortex is shown to assemble a ring-like pattern at the equatorial cortex followed by myosin-driven clustering and consequently blebbing. An active gel theory unifies a model for the obsd. membrane shape changes induced by the contractile cortex.
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  201. 201
    Gardner, P. M.; Winzer, K.; Davis, B. G. Sugar Synthesis in a Protocellular Model Leads to a Cell Signalling Response in Bacteria. Nat. Chem. 2009, 1 (5), 377– 383,  DOI: 10.1038/nchem.296
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    Sugar synthesis in a protocellular model leads to a cell signalling response in bacteria
    Gardner, Paul M.; Winzer, Klaus; Davis, Benjamin G.
    Nature Chemistry (2009), 1 (5), 377-383, S377/1-S377/50CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    The design of systems with life-like properties from simple chem. components may offer insights into biol. processes, with the ultimate goal of creating an artificial chem. cell that would be considered to be alive. Most efforts to create artificial cells have concd. on systems based on complex natural mols. such as DNA and RNA. Here we have constructed a lipid-bound protometabolism that synthesizes complex carbohydrates from simple feedstocks, which are capable of engaging the natural quorum sensing mechanism of the marine bacterium Vibrio harveyi and stimulating a proportional bioluminescent response. This encapsulated system may represent the first step towards the realization of a cellular mimic' and a starting point for bottom-up' designs of other chem. cells, which could perhaps display complex behaviors such as communication with natural cells.
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ACS Synthetic Biology

Cite this: ACS Synth. Biol. 2024, 13, 4, 974–997
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https://doi.org/10.1021/acssynbio.3c00724
Published March 26, 2024

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  • Abstract

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    Figure 1

    Figure 1. Our working definition of living systems, based on Tibor Gánti’s Chemoton model of life. Compartmentalization, replication, and metabolism together make up minimal criteria for a living system. From these other features emerge, including evolution, responsiveness to stimuli, and directed movement.

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    Figure 2

    Figure 2. Four different molecular platforms for studying life. Outgoing from externally provided template DNA (1), a cell-free transcription/translation (Tx/Tl) system uses complex substrates and a chemical energy-donor (2) to synthesize RNA, proteins and potentially other biocatalytically (enzymatically) formed products (3). An encapsulated cell-free system is often a liposome enclosing a cell-free Tx/Tl system. A synthetic cell would function more like a natural cell in that it could be a self-sustaining system that is capable of replication.

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    Figure 3

    Figure 3. Major approaches to building a synthetic cell: bottom-up (a), top-down (b), and middle-out (c) are distinguished as the basic concepts of currently ongoing research. Top-down approaches generally seek to find a “minimal genome” of an existing organism, while the bottom-up approach aspires to de novo create a cell “from scratch” or based on macromolecules. Middle-out approaches utilize modules of known function (e.g., organelles, extracts) to assemble a new cell or major elements/mechanisms thereof.

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    Figure 4

    Figure 4. Milestones toward the de novo design and construction of a synthetic cell. Many of these steps can be performed in parallel with a mixing and matching approach to integration.

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      Trimming the genomic fat: minimising and re-functionalising genomes using synthetic biology
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      Essential genes of a minimal bacterium
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      Mycoplasma genitalium has the smallest genome of any organism that can be grown in pure culture. It has a minimal metab. and little genomic redundancy. Consequently, its genome is expected to be a close approxn. to the minimal set of genes needed to sustain bacterial life. Using global transposon mutagenesis, gene disruption mutants were isolated and characterized for 100 different nonessential protein-coding genes. None of the 43 RNA-coding genes were disrupted. Herein, 382 of the 482 M. genitalium protein-coding genes were identified as essential, plus 5 sets of disrupted genes that encode proteins with potentially redundant essential functions, such as phosphate transport. Genes encoding proteins of unknown function constitute 28% of the essential protein-coding genes set. Disruption of some genes accelerated M. genitalium growth. The genome of M. genitalium G37 (ATCC 33530) was resequenced and found to differ from the previously sequenced version at 34 sites; the new sequences replaces the original M. genitalium genome sequence in GenBank/EMBL/DDBJ under accession no. L43967.
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      27
      Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome
      Hashimoto, Masayuki; Ichimura, Toshiharu; Mizoguchi, Hiroshi; Tanaka, Kimie; Fujimitsu, Kazuyuki; Keyamura, Kenji; Ote, Tomotake; Yamakawa, Takehiro; Yamazaki, Yukiko; Mori, Hideo; Katayama, Tsutomu; Kato, Jun-ichi
      Molecular Microbiology (2005), 55 (1), 137-149CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
      The minimization of a genome is necessary to identify exptl. the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large-scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphol. change. Based on the nucleoid localization obsd. with these mutants, we discuss the cellular nucleoid dynamics.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXotlSrtw%253D%253D&md5=484e3e2ccc942985f12b165903bf23f2
    28. 28
      Pósfai, G.; Plunkett, G., 3rd; Fehér, T.; Frisch, D.; Keil, G. M.; Umenhoffer, K.; Kolisnychenko, V.; Stahl, B.; Sharma, S. S.; de Arruda, M.; Burland, V.; Harcum, S. W.; Blattner, F. R. Emergent Properties of Reduced-Genome Escherichia Coli. Science 2006, 312 (5776), 1044– 1046,  DOI: 10.1126/science.1126439
      There is no corresponding record for this reference.
    29. 29
      Lartigue, C.; Glass, J. I.; Alperovich, N.; Pieper, R.; Parmar, P. P.; Hutchison, C. A., 3rd; Smith, H. O.; Venter, J. C. Genome Transplantation in Bacteria: Changing One Species to Another. Science 2007, 317 (5838), 632– 638,  DOI: 10.1126/science.1144622
      29
      Genome transplantation in bacteria: changing one species to another
      Lartigue, Carole; Glass, John I.; Alperovich, Nina; Pieper, Rembert; Parmar, Prashanth P.; Hutchison, Clyde A., III; Smith, Hamilton O.; Venter, J. Craig
      Science (Washington, DC, United States) (2007), 317 (5838), 632-638CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      As a step toward propagation of synthetic genomes, the authors completely replaced the genome of a bacterial cell with one from another species by transplanting a whole genome as naked DNA. Intact genomic DNA from Mycoplasma mycoides large colony (LC), virtually free of protein, was transplanted into Mycoplasma capricolum cells by polyethylene glycol-mediated transformation. Cells selected for tetracycline resistance, carried by the M. mycoides LC chromosome, contain the complete donor genome and are free of detectable recipient genomic sequences. These cells that result from genome transplantation are phenotypically identical to the M. mycoides LC donor strain as judged by several criteria.
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    30. 30
      Gibson, D. G.; Benders, G. A.; Andrews-Pfannkoch, C.; Denisova, E. A.; Baden-Tillson, H.; Zaveri, J.; Stockwell, T. B.; Brownley, A.; Thomas, D. W.; Algire, M. A.; Merryman, C.; Young, L.; Noskov, V. N.; Glass, J. I.; Venter, J. C.; Hutchison, C. A., 3rd; Smith, H. O. Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma Genitalium Genome. Science 2008, 319 (5867), 1215– 1220,  DOI: 10.1126/science.1151721
      30
      Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome
      Gibson, Daniel G.; Benders, Gwynedd A.; Andrews-Pfannkoch, Cynthia; Denisova, Evgeniya A.; Baden-Tillson, Holly; Zaveri, Jayshree; Stockwell, Timothy B.; Brownley, Anushka; Thomas, David W.; Algire, Mikkel A.; Merryman, Chuck; Young, Lei; Noskov, Vladimir N.; Glass, John I.; Venter, J. Craig; Hutchison, Clyde A., III; Smith, Hamilton O.
      Science (Washington, DC, United States) (2008), 319 (5867), 1215-1220CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chem. synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approx. 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-assocd. recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA mols. from chem. synthesized pieces and also from combinations of natural and synthetic DNA segments.
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    31. 31
      Lartigue, C.; Vashee, S.; Algire, M. A.; Chuang, R.-Y.; Benders, G. A.; Ma, L.; Noskov, V. N.; Denisova, E. A.; Gibson, D. G.; Assad-Garcia, N.; Alperovich, N.; Thomas, D. W.; Merryman, C.; Hutchison, C. A., 3rd; Smith, H. O.; Venter, J. C.; Glass, J. I. Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast. Science 2009, 325 (5948), 1693– 1696,  DOI: 10.1126/science.1173759
      31
      Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast
      Lartigue, Carole; Vashee, Sanjay; Algire, Mikkel A.; Chuang, Ray-Yuan; Benders, Gwynedd A.; Ma, Li; Noskov, Vladimir N.; Denisova, Evgeniya A.; Gibson, Daniel G.; Assad-Garcia, Nacyra; Alperovich, Nina; Thomas, David W.; Merryman, Chuck; Hutchison, Clyde A., III; Smith, Hamilton O.; Venter, J. Craig; Glass, John I.
      Science (Washington, DC, United States) (2009), 325 (5948), 1693-1696CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      We recently reported the chem. synthesis, assembly, and cloning of a bacterial genome in yeast. To produce a synthetic cell, the genome must be transferred from yeast to a receptive cytoplasm. Here we describe methods to accomplish this. We cloned a Mycoplasma mycoides genome as a yeast centromeric plasmid and then transplanted it into Mycoplasma capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast genetic systems and then transplanted to produce a new strain of M. mycoides. These methods allow the construction of strains that could not be produced with genetic tools available for this bacterium. The complete, annotated sequence of the transplanted M. mycoides capri genome is deposited in GenBank/EMBL/DDBJ with accession no. CP001668.
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    32. 32
      Gibson, D. G.; Glass, J. I.; Lartigue, C.; Noskov, V. N.; Chuang, R.-Y.; Algire, M. A.; Benders, G. A.; Montague, M. G.; Ma, L.; Moodie, M. M.; Merryman, C.; Vashee, S.; Krishnakumar, R.; Assad-Garcia, N.; Andrews-Pfannkoch, C.; Denisova, E. A.; Young, L.; Qi, Z.-Q.; Segall-Shapiro, T. H.; Calvey, C. H.; Parmar, P. P.; Hutchison, C. A., 3rd; Smith, H. O.; Venter, J. C. Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science 2010, 329 (5987), 52– 56,  DOI: 10.1126/science.1190719
      32
      Creation of a bacterial cell controlled by a chemically synthesized genome
      Gibson, Daniel G.; Glass, John I.; Lartigue, Carole; Noskov, Vladimir N.; Chuang, Ray-Yuan; Algire, Mikkel A.; Benders, Gwynedd A.; Montague, Michael G.; Ma, Li; Moodie, Monzia M.; Merryman, Chuck; Vashee, Sanjay; Krishnakumar, Radha; Assad-Garcia, Nacyra; Andrews-Pfannkoch, Cynthia; Denisova, Evgeniya A.; Young, Lei; Qi, Zhi-Qing; Segall-Shapiro, Thomas H.; Calvey, Christopher H.; Parmar, Prashanth P.; Hutchison, Clyde A., III; Smith, Hamilton O.; Venter, J. Craig
      Science (Washington, DC, United States) (2010), 329 (5987), 52-56CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication. The complete, annotated synthetic genome sequence is deposited in GenBank/EMBL/DDBJ with accession no. CP002027.
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    33. 33
      Stano, P.; Luisi, P. L. Semi-Synthetic Minimal Cells: Origin and Recent Developments. Curr. Opin. Biotechnol. 2013, 24 (4), 633– 638,  DOI: 10.1016/j.copbio.2013.01.002
      33
      Semi-synthetic minimal cells: origin and recent developments
      Stano, Pasquale; Luisi, Pier Luigi
      Current Opinion in Biotechnology (2013), 24 (4), 633-638CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)
      A review. The notion of minimal cells refers to cellular structures that contain the minimal and sufficient complexity to still be defined as living, or at least capable to display the most important features of biol. cells. Here the authors briefly describe the lab. construction of minimal cells, a project within the broader field of synthetic biol. In particular the authors discuss the advancements in the prepn. of semi-synthetic cells based on the encapsulation of biochems. inside liposomes, illustrating from the one hand the origin of this research and the most recent developments; and from the other the difficulties and limits of the approach. The role of physicochem. understandings is greatly emphasized.
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    34. 34
      Olivi, L.; Berger, M.; Creyghton, R. N. P.; De Franceschi, N.; Dekker, C.; Mulder, B. M.; Claassens, N. J.; Ten Wolde, P. R.; van der Oost, J. Towards a Synthetic Cell Cycle. Nat. Commun. 2021, 12 (1), 4531,  DOI: 10.1038/s41467-021-24772-8
      34
      Towards a synthetic cell cycle
      Olivi, Lorenzo; Berger, Mareike; Creyghton, Ramon N. P.; De Franceschi, Nicola; Dekker, Cees; Mulder, Bela M.; Claassens, Nico J.; ten Wolde, Pieter Rein; van der Oost, John
      Nature Communications (2021), 12 (1), 4531CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: Recent developments in synthetic biol. may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. Here, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this Perspective, we discuss potential paths towards an integrated synthetic cell cycle.
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    35. 35
      Adamala, K. P.; Martin-Alarcon, D. A.; Guthrie-Honea, K. R.; Boyden, E. S. Engineering Genetic Circuit Interactions within and between Synthetic Minimal Cells. Nat. Chem. 2017, 9 (5), 431– 439,  DOI: 10.1038/nchem.2644
      35
      Engineering genetic circuit interactions within and between synthetic minimal cells
      Adamala, Katarzyna P.; Martin-Alarcon, Daniel A.; Guthrie-Honea, Katriona R.; Boyden, Edward S.
      Nature Chemistry (2017), 9 (5), 431-439CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      Genetic circuits and reaction cascades are of great importance for synthetic biol., biochem. and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chem. reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-contg. synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biol. cascades, an essential step towards their ultimate programmability.
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    36. 36
      Fenz, S. F.; Sachse, R.; Schmidt, T.; Kubick, S. Cell-Free Synthesis of Membrane Proteins: Tailored Cell Models out of Microsomes. Biochim. Biophys. Acta 2014, 1838 (5), 1382– 1388,  DOI: 10.1016/j.bbamem.2013.12.009
      There is no corresponding record for this reference.
    37. 37
      Kroll, A. V.; Fang, R. H.; Zhang, L. Biointerfacing and Applications of Cell Membrane-Coated Nanoparticles. Bioconjugate Chem. 2017, 28 (1), 23– 32,  DOI: 10.1021/acs.bioconjchem.6b00569
      37
      Biointerfacing and Applications of Cell Membrane-Coated Nanoparticles
      Kroll, Ashley V.; Fang, Ronnie H.; Zhang, Liangfang
      Bioconjugate Chemistry (2017), 28 (1), 23-32CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      The cell membrane-coated nanoparticle is a biomimetic platform consisting of a nanoparticulate core coated with membrane derived from a cell, such as a red blood cell, platelet, or cancer cell. The cell membrane "disguise" allows the particles to be perceived by the body as the source cell by interacting with its surroundings using the translocated surface membrane components. The newly bestowed characteristics of the membrane-coated nanoparticle can be utilized for biol. interfacing in the body, providing natural solns. to many biomedical issues. This Review will cover the interactions of these cell membrane-coated nanoparticles and their applications within three biomedical areas of interest, including (i) drug delivery, (ii) detoxification, and (iii) immune modulation.
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    38. 38
      Gurramkonda, C.; Rao, A.; Borhani, S.; Pilli, M.; Deldari, S.; Ge, X.; Pezeshk, N.; Han, T.-C.; Tolosa, M.; Kostov, Y.; Tolosa, L.; Wood, D. W.; Vattem, K.; Frey, D. D.; Rao, G. Improving the Recombinant Human Erythropoietin Glycosylation Using Microsome Supplementation in CHO Cell-Free System. Biotechnol. Bioeng. 2018, 115 (5), 1253– 1264,  DOI: 10.1002/bit.26554
      There is no corresponding record for this reference.
    39. 39
      Su’etsugu, M.; Takada, H.; Katayama, T.; Tsujimoto, H. Exponential Propagation of Large Circular DNA by Reconstitution of a Chromosome-Replication Cycle. Nucleic Acids Res. 2017, 45 (20), 11525– 11534,  DOI: 10.1093/nar/gkx822
      39
      Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle
      Su'etsugu, Masayuki; Takada, Hiraku; Katayama, Tsutomu; Tsujimoto, Hiroko
      Nucleic Acids Research (2017), 45 (20), 11525-11534CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      Propagation of genetic information is a fundamental property of living organisms. Escherichia coli has a 4.6 Mb circular chromosome with a replication origin, oriC. While the oriC replication has been reconstituted in vitro more than 30 years ago, continuous repetition of the replication cycle has not yet been achieved. Here, we reconstituted the entire replication cycle with 14 purified enzymes (25 polypeptides) that catalyze initiation at oriC, bidirectional fork progression, Okazaki-fragment maturation and decatenation of the replicated circular products. Because decatenation provides covalently closed supercoiled monomers that are competent for the next round of replication initiation, the replication cycle repeats autonomously and continuously in an isothermal condition. This replication-cycle reaction (RCR) propagates ∼10 kb circular DNA exponentially as intact covalently closed mols., even from a single DNA mol., with a doubling time of ∼8 min and extremely high fidelity. Very large DNA up to 0.2 Mb is successfully propagated within 3 h. We further demonstrate a cell-free cloning in which RCR selectively propagates circular mols. constructed by a multi-fragment assembly reaction. Our results define the min. element necessary for the repetition of the chromosome-replication cycle, and also provide a powerful in vitro tool to generate large circular DNA mols. without relying on conventional biol. cloning.
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    40. 40
      Shimizu, Y.; Inoue, A.; Tomari, Y.; Suzuki, T.; Yokogawa, T.; Nishikawa, K.; Ueda, T. Cell-Free Translation Reconstituted with Purified Components. Nat. Biotechnol. 2001, 19 (8), 751– 755,  DOI: 10.1038/90802
      40
      Cell-free translation reconstituted with purified components
      Shimizu, Yoshihiro; Inoue, Akio; Tomari, Yukihide; Suzuki, Tsutomu; Yokogawa, Takashi; Nishikawa, Kazuya; Ueda, Takuya
      Nature Biotechnology (2001), 19 (8), 751-755CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
      We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 μg/mL/h in a batch mode without the need for any supplementary app. The protein products were easily purified within 1 h using affinity chromatog. to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor tRNA.
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    41. 41
      Drienovská, I.; Roelfes, G. Expanding the Enzyme Universe with Genetically Encoded Unnatural Amino Acids. Nature Catalysis 2020, 3 (3), 193– 202,  DOI: 10.1038/s41929-019-0410-8
      There is no corresponding record for this reference.
    42. 42
      Medina, E.; Yik, E. J.; Herdewijn, P.; Chaput, J. C. Functional Comparison of Laboratory-Evolved XNA Polymerases for Synthetic Biology. ACS Synth. Biol. 2021, 10 (6), 1429– 1437,  DOI: 10.1021/acssynbio.1c00048
      There is no corresponding record for this reference.
    43. 43
      Theobald, D. L. A Formal Test of the Theory of Universal Common Ancestry. Nature 2010, 465 (7295), 219– 222,  DOI: 10.1038/nature09014
      There is no corresponding record for this reference.
    44. 44
      Yang, Z.; Chen, F.; Alvarado, J. B.; Benner, S. A. Amplification, Mutation, and Sequencing of a Six-Letter Synthetic Genetic System. J. Am. Chem. Soc. 2011, 133 (38), 15105– 15112,  DOI: 10.1021/ja204910n
      44
      Amplification, mutation, and sequencing of a six-letter synthetic genetic system
      Yang, Zun-Yi; Chen, Fei; Alvarado, J. Brian; Benner, Steven A.
      Journal of the American Chemical Society (2011), 133 (38), 15105-15112CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The next goals in the development of a synthetic biol. that uses artificial genetic systems will require chem.-biol. combinations that allow the amplification of DNA contg. any no. of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technol. is needed to sequence artificial genetic DNA mols. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four std. nucleotides (G, A, C, and T) and two addnl. nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theor. cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.
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    45. 45
      Malyshev, D. A.; Dhami, K.; Lavergne, T.; Chen, T.; Dai, N.; Foster, J. M.; Corrêa, I. R., Jr; Romesberg, F. E. A Semi-Synthetic Organism with an Expanded Genetic Alphabet. Nature 2014, 509 (7500), 385– 388,  DOI: 10.1038/nature13314
      45
      A semi-synthetic organism with an expanded genetic alphabet
      Malyshev, Denis A.; Dhami, Kirandeep; Lavergne, Thomas; Chen, Tingjian; Dai, Nan; Foster, Jeremy M.; Correa, Ivan R.; Romesberg, Floyd E.
      Nature (London, United Kingdom) (2014), 509 (7500), 385-388CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA contg. the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid contg. d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
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    46. 46
      Rech, E. L.; Arber, W. Biodiversity as a Source for Synthetic Domestication of Useful Specific Traits. Ann. Appl. Biol. 2013, 162 (2), 141– 144,  DOI: 10.1111/aab.12013
      There is no corresponding record for this reference.
    47. 47
      Rech, E. Genomics and Synthetic Biology as a Viable Option to Intensify Sustainable Use of Biodiversity. Nat. Precedings 2011, 1– 1,  DOI: 10.1038/npre.2011.5759.1
      There is no corresponding record for this reference.
    48. 48
      Kiyama, H.; Kakizawa, S.; Sasajima, Y.; Tahara, Y. O.; Miyata, M. Reconstitution of a Minimal Motility System Based on Spiroplasma Swimming by Two Bacterial Actins in a Synthetic Minimal Bacterium. Sci. Adv. 2022, 8 (48), eabo7490,  DOI: 10.1126/sciadv.abo7490
      There is no corresponding record for this reference.
    49. 49
      Geiger, O.; Sanchez-Flores, A.; Padilla-Gomez, J.; Degli Esposti, M. Multiple Approaches of Cellular Metabolism Define the Bacterial Ancestry of Mitochondria. Sci. Adv. 2023, 9 (32), eadh0066,  DOI: 10.1126/sciadv.adh0066
      There is no corresponding record for this reference.
    50. 50
      Pinheiro, V. B.; Taylor, A. I.; Cozens, C.; Abramov, M.; Renders, M.; Zhang, S.; Chaput, J. C.; Wengel, J.; Peak-Chew, S.-Y.; McLaughlin, S. H.; Herdewijn, P.; Holliger, P. Synthetic Genetic Polymers Capable of Heredity and Evolution. Science 2012, 336 (6079), 341– 344,  DOI: 10.1126/science.1217622
      50
      Synthetic Genetic Polymers Capable of Heredity and Evolution
      Pinheiro, Vitor B.; Taylor, Alexander I.; Cozens, Christopher; Abramov, Mikhail; Renders, Marleen; Zhang, Su; Chaput, John C.; Wengel, Jesper; Peak-Chew, Sew-Yeu; McLaughlin, Stephen H.; Herdewijn, Piet; Holliger, Philipp
      Science (Washington, DC, United States) (2012), 336 (6079), 341-344CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Genetic information storage and processing rely on just two polymers, DNA and RNA, yet whether their role reflects evolutionary history or fundamental functional constraints is currently unknown. With the use of polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers based on simple nucleic acid architectures not found in nature [xeno-nucleic acids (XNAs)]. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and folding into defined structures. Thus, heredity and evolution, two hallmarks of life, are not limited to DNA and RNA but are likely to be emergent properties of polymers capable of information storage.
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    51. 51
      Hoshika, S.; Leal, N. A.; Kim, M.-J.; Kim, M.-S.; Karalkar, N. B.; Kim, H.-J.; Bates, A. M.; Watkins, N. E., Jr; SantaLucia, H. A.; Meyer, A. J.; DasGupta, S.; Piccirilli, J. A.; Ellington, A. D.; SantaLucia, J., Jr; Georgiadis, M. M.; Benner, S. A. Hachimoji DNA and RNA: A Genetic System with Eight Building Blocks. Science 2019, 363 (6429), 884– 887,  DOI: 10.1126/science.aat0971
      51
      Hachimoji DNA and RNA: A genetic system with eight building blocks
      Hoshika, Shuichi; Leal, Nicole A.; Kim, Myong-Jung; Kim, Myong-Sang; Karalkar, Nilesh B.; Kim, Hyo-Joong; Bates, Alison M.; Watkins, Norman E., Jr.; SantaLucia, Holly A.; Meyer, Adam J.; DasGupta, Saurja; Piccirilli, Joseph A.; Ellington, Andrew D.; SantaLucia, John, Jr.; Georgiadis, Millie M.; Benner, Steven A.
      Science (Washington, DC, United States) (2019), 363 (6429), 884-887CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodn. stability, and stereoregular building blocks that fit a Schroedinger aperiodic crystal. Measured thermodn. parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information d. of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of mol. structures that might support life, including life throughout the cosmos.
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    52. 52
      Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; Bridgland, A.; Meyer, C.; Kohl, S. A. A.; Ballard, A. J.; Cowie, A.; Romera-Paredes, B.; Nikolov, S.; Jain, R.; Adler, J.; Back, T.; Petersen, S.; Reiman, D.; Clancy, E.; Zielinski, M.; Steinegger, M.; Pacholska, M.; Berghammer, T.; Bodenstein, S.; Silver, D.; Vinyals, O.; Senior, A. W.; Kavukcuoglu, K.; Kohli, P.; Hassabis, D. Highly Accurate Protein Structure Prediction with AlphaFold. Nature 2021, 596 (7873), 583– 589,  DOI: 10.1038/s41586-021-03819-2
      52
      Highly accurate protein structure prediction with AlphaFold
      Jumper, John; Evans, Richard; Pritzel, Alexander; Green, Tim; Figurnov, Michael; Ronneberger, Olaf; Tunyasuvunakool, Kathryn; Bates, Russ; Zidek, Augustin; Potapenko, Anna; Bridgland, Alex; Meyer, Clemens; Kohl, Simon A. A.; Ballard, Andrew J.; Cowie, Andrew; Romera-Paredes, Bernardino; Nikolov, Stanislav; Jain, Rishub; Adler, Jonas; Back, Trevor; Petersen, Stig; Reiman, David; Clancy, Ellen; Zielinski, Michal; Steinegger, Martin; Pacholska, Michalina; Berghammer, Tamas; Bodenstein, Sebastian; Silver, David; Vinyals, Oriol; Senior, Andrew W.; Kavukcuoglu, Koray; Kohli, Pushmeet; Hassabis, Demis
      Nature (London, United Kingdom) (2021), 596 (7873), 583-589CODEN: NATUAS; ISSN:0028-0836. (Nature Portfolio)
      Proteins are essential to life, and understanding their structure can facilitate a mechanistic understanding of their function. Through an enormous exptl. effort, the structures of around 100,000 unique proteins have been detd., but this represents a small fraction of the billions of known protein sequences. Structural coverage is bottlenecked by the months to years of painstaking effort required to det. a single protein structure. Accurate computational approaches are needed to address this gap and to enable large-scale structural bioinformatics. Predicting the three-dimensional structure that a protein will adopt based solely on its amino acid sequence-the structure prediction component of the 'protein folding problem'-has been an important open research problem for more than 50 years. Despite recent progress, existing methods fall far short of at. accuracy, esp. when no homologous structure is available. Here we provide the first computational method that can regularly predict protein structures with at. accuracy even in cases in which no similar structure is known. We validated an entirely redesigned version of our neural network-based model, AlphaFold, in the challenging 14th Crit. Assessment of protein Structure Prediction (CASP14), demonstrating accuracy competitive with exptl. structures in a majority of cases and greatly outperforming other methods. Underpinning the latest version of AlphaFold is a novel machine learning approach that incorporates phys. and biol. knowledge about protein structure, leveraging multi-sequence alignments, into the design of the deep learning algorithm.
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    53. 53
      Lee, J. W.; Na, D.; Park, J. M.; Lee, J.; Choi, S.; Lee, S. Y. Systems Metabolic Engineering of Microorganisms for Natural and Non-Natural Chemicals. Nat. Chem. Biol. 2012, 8 (6), 536– 546,  DOI: 10.1038/nchembio.970
      53
      Systems metabolic engineering of microorganisms for natural and non-natural chemicals
      Lee, Jeong Wook; Na, Dokyun; Park, Jong Myoung; Lee, Joungmin; Choi, Sol; Lee, Sang Yup
      Nature Chemical Biology (2012), 8 (6), 536-546CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      A review. Growing concerns over limited fossil resources and assocd. environmental problems are motivating the development of sustainable processes for the prodn. of chems., fuels and materials from renewable resources. Metabolic engineering is a key enabling technol. for transforming microorganisms into efficient cell factories for these compds. Systems metabolic engineering, which incorporates the concepts and techniques of systems biol., synthetic biol. and evolutionary engineering at the systems level, offers a conceptual and technol. framework to speed the creation of new metabolic enzymes and pathways or the modification of existing pathways for the optimal prodn. of desired products. Here we discuss the general strategies of systems metabolic engineering and examples of its application and offer insights as to when and how each of the different strategies should be used. Finally, we highlight the limitations and challenges to be overcome for the systems metabolic engineering of microorganisms at more advanced levels.
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    54. 54
      Nielsen, J. Systems Biology of Metabolism. Annu. Rev. Biochem. 2017, 86, 245– 275,  DOI: 10.1146/annurev-biochem-061516-044757
      54
      Systems Biology of Metabolism
      Nielsen, Jens
      Annual Review of Biochemistry (2017), 86 (), 245-275CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
      Metab. is highly complex and involves thousands of different connected reactions; it is therefore necessary to use math. models for holistic studies. The use of math. models in biol. is referred to as systems biol. In this review, the principles of systems biol. are described, and two different types of math. models used for studying metab. are discussed: kinetic models and genome-scale metabolic models. The use of different omics technologies, including transcriptomics, proteomics, metabolomics, and fluxomics, for studying metab. is presented. Finally, the application of systems biol. for analyzing global regulatory structures, engineering the metab. of cell factories, and analyzing human diseases is discussed.
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    55. 55
      Choi, K. R.; Jang, W. D.; Yang, D.; Cho, J. S.; Park, D.; Lee, S. Y. Systems Metabolic Engineering Strategies: Integrating Systems and Synthetic Biology with Metabolic Engineering. Trends Biotechnol. 2019, 37 (8), 817– 837,  DOI: 10.1016/j.tibtech.2019.01.003
      55
      Systems Metabolic Engineering Strategies: Integrating Systems and Synthetic Biology with Metabolic Engineering
      Choi, Kyeong Rok; Jang, Woo Dae; Yang, Dongsoo; Cho, Jae Sung; Park, Dahyeon; Lee, Sang Yup
      Trends in Biotechnology (2019), 37 (8), 817-837CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
      A review. Metabolic engineering allows development of microbial strains efficiently producing chems. and materials, but it requires much time, effort, and cost to make the strains industrially competitive. Systems metabolic engineering, which integrates tools and strategies of systems biol., synthetic biol., and evolutionary engineering with traditional metabolic engineering, has recently been used to facilitate development of high-performance strains. The past decade has witnessed this interdisciplinary strategy continuously being improved toward the development of industrially competitive overproducer strains. In this article, current trends in systems metabolic engineering including tools and strategies are reviewed, focusing on recent developments in selection of host strains, metabolic pathway reconstruction, tolerance enhancement, and metabolic flux optimization. Also, future challenges and prospects are discussed.
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    56. 56
      Karim, A. S.; Dudley, Q. M.; Juminaga, A.; Yuan, Y.; Crowe, S. A.; Heggestad, J. T.; Garg, S.; Abdalla, T.; Grubbe, W. S.; Rasor, B. J.; Coar, D. N.; Torculas, M.; Krein, M.; Liew, F. E.; Quattlebaum, A.; Jensen, R. O.; Stuart, J. A.; Simpson, S. D.; Köpke, M.; Jewett, M. C. In Vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes for Cell Design. Nat. Chem. Biol. 2020, 16 (8), 912– 919,  DOI: 10.1038/s41589-020-0559-0
      56
      In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design
      Karim, Ashty S.; Dudley, Quentin M.; Juminaga, Alex; Yuan, Yongbo; Crowe, Samantha A.; Heggestad, Jacob T.; Garg, Shivani; Abdalla, Tanus; Grubbe, William S.; Rasor, Blake J.; Coar, David N.; Torculas, Maria; Krein, Michael; Liew, FungMin; Quattlebaum, Amy; Jensen, Rasmus O.; Stuart, Jeffrey A.; Simpson, Sean D.; Kopke, Michael; Jewett, Michael C.
      Nature Chemical Biology (2020), 16 (8), 912-919CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
      The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced nos. of validated genetic parts and a lack of high-throughput workflows. Here the authors describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. The authors demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate prodn. and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, the authors then scaled up the authors' highest-performing pathway, which improved in vivo 3-HB prodn. in Clostridium by 20-fold to 14.63 ± 0.48 g L-1. The authors expect iPROBE to accelerate design-build-test cycles for industrial biotechnol.
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    57. 57
      Dudley, Q. M.; Karim, A. S.; Nash, C. J.; Jewett, M. C. In Vitro Prototyping of Limonene Biosynthesis Using Cell-Free Protein Synthesis. Metab. Eng. 2020, 61, 251– 260,  DOI: 10.1016/j.ymben.2020.05.006
      57
      In vitro prototyping of limonene biosynthesis using cell-free protein synthesis
      Dudley, Quentin M.; Karim, Ashty S.; Nash, Connor J.; Jewett, Michael C.
      Metabolic Engineering (2020), 61 (), 251-260CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)
      Metabolic engineering of microorganisms to produce sustainable chems. has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-build-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large no. of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manuf. pathway enzymes in sep. reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in exts. from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concns. on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene prodn. in 24 h from 0.2 to 4.5 mM (23-610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.
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    58. 58
      Averesch, N. J. H.; Krömer, J. O. Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds-Present and Future Strain Construction Strategies. Front. Bioeng. Biotechnol. 2018, 6, 32,  DOI: 10.3389/fbioe.2018.00032
      58
      Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds-Present and Future Strain Construction Strategies
      Averesch Nils J H; Kromer Jens O
      Frontiers in bioengineering and biotechnology (2018), 6 (), 32 ISSN:2296-4185.
      The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Re)construction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations.
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    59. 59
      Jang, W. D.; Kim, G. B.; Lee, S. Y. An Interactive Metabolic Map of Bio-Based Chemicals. Trends Biotechnol. 2023, 41 (1), 10– 14,  DOI: 10.1016/j.tibtech.2022.07.013
      There is no corresponding record for this reference.
    60. 60
      Lai, H.-E.; Obled, A. M. C.; Chee, S. M.; Morgan, R. M.; Lynch, R.; Sharma, S. V.; Moore, S. J.; Polizzi, K. M.; Goss, R. J. M.; Freemont, P. S. GenoChemetic Strategy for Derivatization of the Violacein Natural Product Scaffold. ACS Chem. Biol. 2021, 16 (11), 2116– 2123,  DOI: 10.1021/acschembio.1c00483
      60
      GenoChemetic Strategy for Derivatization of the Violacein Natural Product Scaffold
      Lai, Hung-En; Obled, Alan M. C.; Chee, Soo Mei; Morgan, Rhodri M.; Lynch, Rosemary; Sharma, Sunil V.; Moore, Simon J.; Polizzi, Karen M.; Goss, Rebecca J. M.; Freemont, Paul S.
      ACS Chemical Biology (2021), 16 (11), 2116-2123CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Natural products and their analogs are often challenging to synthesize due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compd. diversity. Integrating synthetic biol. with synthetic chem. allows rapid access to much more diverse portfolios of xenobiotic compds., which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, six halogenated analogs of violacein or deoxyviolacein were generated, demonstrating the promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivs. were generated from 5-brominated violacein analogs via the Suzuki-Miyaura cross-coupling reaction directly using the crude ext. without prior purifn. Herein we demonstrate a flexible and rapid approach to access a diverse chem. space that can be applied to a wide range of natural product scaffolds.
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    61. 61
      Galanie, S.; Thodey, K.; Trenchard, I. J.; Filsinger Interrante, M.; Smolke, C. D. Complete Biosynthesis of Opioids in Yeast. Science 2015, 349 (6252), 1095– 1100,  DOI: 10.1126/science.aac9373
      61
      Complete biosynthesis of opioids in yeast
      Galanie, Stephanie; Thodey, Kate; Trenchard, Isis J.; Filsinger Interrante, Maria; Smolke, Christina D.
      Science (Washington, DC, United States) (2015), 349 (6252), 1095-1100CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Opioids are the primary drugs used in Western medicine for pain management and palliative care. Farming of opium poppies remains the sole source of these essential medicines, despite diverse market demands and uncertainty in crop yields due to weather, climate change, and pests. We engineered yeast to produce the selected opioid compds. thebaine and hydrocodone starting from sugar. All work was conducted in a lab. that is permitted and secured for work with controlled substances. We combined enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast. The resulting opioid biosynthesis strains required the expression of 21 (thebaine) and 23 (hydrocodone) enzyme activities from plants, mammals, bacteria, and yeast itself. This is a proof of principle, and major hurdles remain before optimization and scale-up could be achieved. Open discussions of options for governing this technol. are also needed in order to responsibly realize alternative supplies for these medically relevant compds.
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    62. 62
      Trenchard, I. J.; Siddiqui, M. S.; Thodey, K.; Smolke, C. D. De Novo Production of the Key Branch Point Benzylisoquinoline Alkaloid Reticuline in Yeast. Metab. Eng. 2015, 31, 74– 83,  DOI: 10.1016/j.ymben.2015.06.010
      62
      De novo production of the key branch point benzylisoquinoline alkaloid reticuline in yeast
      Trenchard, Isis J.; Siddiqui, Michael S.; Thodey, Kate; Smolke, Christina D.
      Metabolic Engineering (2015), 31 (), 74-83CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)
      Microbial biosynthesis for plant-based natural products, such as the benzylisoquinoline alkaloids (BIAs), has the potential to address limitations in plant-based supply of established drugs and make new mols. available for drug discovery. While yeast strains have been engineered to produce a variety of downstream BIAs including the opioids, these strains have relied on feeding an early BIA substrate. We describe the de novo synthesis of the major BIA branch point intermediate reticuline via norcoclaurine in Saccharomyces cerevisiae. Modifications were introduced into yeast central metab. to increase supply of the BIA precursor tyrosine, allowing us to achieve a 60-fold increase in prodn. of the early benzylisoquinoline scaffold from fed dopamine with no supply of exogenous tyrosine. Yeast strains further engineered to express a mammalian tyrosine hydroxylase, 4 mammalian tetrahydrobiopterin biosynthesis and recycling enzymes, and a bacterial DOPA decarboxylase produced norcoclaurine de novo. We further increased prodn. of early benzylisoquinoline scaffolds by 160-fold through introducing mutant tyrosine hydroxylase enzymes, an optimized plant norcoclaurine synthase variant, and optimizing culture conditions. Finally, we incorporated 5 addnl. plant enzymes (3 methyltransferases, a cytochrome P 450, and its reductase partner) to achieve de novo prodn. of the key branch point mol. reticuline with a titer of 19.2 μg/L. These strains and reconstructed pathways will serve as a platform for the biosynthesis of diverse natural and novel BIAs.
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    63. 63
      Chan, C. T. Y.; Lee, J. W.; Cameron, D. E.; Bashor, C. J.; Collins, J. J. Deadman” and “Passcode” Microbial Kill Switches for Bacterial Containment. Nat. Chem. Biol. 2016, 12 (2), 82– 86,  DOI: 10.1038/nchembio.1979
      63
      'Deadman' and 'Passcode' microbial kill switches for bacterial containment
      Chan, Clement T. Y.; Lee, Jeong Wook; Cameron, D. Ewen; Bashor, Caleb J.; Collins, James J.
      Nature Chemical Biology (2016), 12 (2), 82-86CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safeguard systems known as the 'Deadman' and 'Passcode' kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI-GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFemsbrE&md5=c1cd6452791fc321b2432e61ad682557
    64. 64
      Caliando, B. J.; Voigt, C. A. Targeted DNA Degradation Using a CRISPR Device Stably Carried in the Host Genome. Nat. Commun. 2015, 6, 6989,  DOI: 10.1038/ncomms7989
      64
      Targeted DNA degradation using a CRISPR device stably carried in the host genome
      Caliando, Brian J.; Voigt, Christopher A.
      Nature Communications (2015), 6 (), 6989CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      Once an engineered organism completes its task, it is useful to degrade the assocd. DNA to reduce environmental release and protect intellectual property. Here we present a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA. This enables engineered regions to be obscured when the cell enters a new environment. DNAi is based on type-IE CRISPR biochem. and a synthetic CRISPR array defines the DNA target(s). When the input is on, plasmid DNA is degraded 108-fold. When the genome is targeted, this causes cell death, reducing viable cells by a factor of 108. Further, the CRISPR nuclease can direct degrdn. to specific genomic regions (for example, engineered or inserted DNA), which could be used to complicate recovery and sequencing efforts. DNAi can be stably carried in an engineered organism, with no impact on cell growth, plasmid stability or DNAi inducibility even after passaging for >2 mo.
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    65. 65
      Nyerges, A.; Vinke, S.; Flynn, R.; Owen, S. V.; Rand, E. A.; Budnik, B.; Keen, E.; Narasimhan, K.; Marchand, J. A.; Baas-Thomas, M.; Liu, M.; Chen, K.; Chiappino-Pepe, A.; Hu, F.; Baym, M.; Church, G. M. A Swapped Genetic Code Prevents Viral Infections and Gene Transfer. Nature 2023, 615 (7953), 720– 727,  DOI: 10.1038/s41586-023-05824-z
      There is no corresponding record for this reference.
    66. 66
      Scrinis, G.; Lyons, K. The Emerging Nano-Corporate Paradigm: Nanotechnology and the Transformation of Nature, Food and Agri-Food Systems. Int. J. Sociol. Agric. Food 2007, 15 (2), 22– 44
      There is no corresponding record for this reference.
    67. 67
      Nitrogen fixing bacteria - microbial fertilizer. https://www.pivotbio.com/ (accessed on April 30, 2023).
      There is no corresponding record for this reference.
    68. 68
      Kaul, S.; Choudhary, M.; Gupta, S.; Dhar, M. K. Engineering Host Microbiome for Crop Improvement and Sustainable Agriculture. Front. Microbiol. 2021, 12, 635917,  DOI: 10.3389/fmicb.2021.635917
      There is no corresponding record for this reference.
    69. 69
      Mueller, U. G.; Sachs, J. L. Engineering Microbiomes to Improve Plant and Animal Health. Trends Microbiol. 2015, 23 (10), 606– 617,  DOI: 10.1016/j.tim.2015.07.009
      69
      Engineering Microbiomes to Improve Plant and Animal Health
      Mueller, U. G.; Sachs, J. L.
      Trends in Microbiology (2015), 23 (10), 606-617CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
      A review. Animal and plant microbiomes encompass diverse microbial communities that colonize every accessible host tissue. These microbiomes enhance host functions, contributing to host health and fitness. A novel approach to improve animal and plant fitness is to artificially select upon microbiomes, thus engineering evolved microbiomes with specific effects on host fitness. The authors call this engineering approach host-mediated microbiome selection, because this method selects upon microbial communities indirectly through the host and leverages host traits that evolved to influence microbiomes. In essence, host phenotypes were used as probes to gauge and manipulate those microbiome functions that impact host fitness. To facilitate research on host-mediated microbiome engineering, the authors explain and compare the principal methods to impose artificial selection on microbiomes; discuss advantages and potential challenges of each method; offer a skeptical appraisal of each method in light of these potential challenges; and outline exptl. strategies to optimize microbiome engineering. Finally, the authors develop a predictive framework for microbiome engineering that organizes research around principles of artificial selection, quant. genetics, and microbial community ecol.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFKhtL7L&md5=8743f515980f584435a175d2b74a33bd
    70. 70
      Jin Song, S.; Woodhams, D. C.; Martino, C.; Allaband, C.; Mu, A.; Javorschi-Miller-Montgomery, S.; Suchodolski, J. S.; Knight, R. Engineering the Microbiome for Animal Health and Conservation. Exp. Biol. Med. 2019, 244 (6), 494– 504,  DOI: 10.1177/1535370219830075
      There is no corresponding record for this reference.
    71. 71
      Aziz, C. E.; Borden, R. C.; Coates, J. D.; Cox, E. E.; Downey, D. C.; Evans, P. J.; Hatzinger, P. B.; Henry, B. M.; Andrew Jackson, W.; Krug, T. A.; Tony Lieberman, M.; Loehr, R. C.; Norris, R. D.; Nzengung, V. A.; Perlmutter, M. W.; Schaefer, C. E.; Stroo, H. F.; Herb Ward, C.; Winstead, C. J.; Wolfe, C. In Situ Bioremediation of Perchlorate in Groundwater; Springer: New York.
      There is no corresponding record for this reference.
    72. 72
      Hou, D.; O’Connor, D.; Igalavithana, A. D.; Alessi, D. S.; Luo, J.; Tsang, D. C. W.; Sparks, D. L.; Yamauchi, Y.; Rinklebe, J.; Ok, Y. S. Metal Contamination and Bioremediation of Agricultural Soils for Food Safety and Sustainability. Nature Reviews Earth & Environment 2020, 1 (7), 366– 381,  DOI: 10.1038/s43017-020-0061-y
      There is no corresponding record for this reference.
    73. 73
      Maity, W.; Maity, S.; Bera, S.; Roy, A. Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes. Appl. Biochem. Biotechnol. 2021, 193 (8), 2699– 2716,  DOI: 10.1007/s12010-021-03562-4
      There is no corresponding record for this reference.
    74. 74
      de Lorenzo, V.; Prather, K. L.; Chen, G.-Q.; O’Day, E.; von Kameke, C.; Oyarzún, D. A.; Hosta-Rigau, L.; Alsafar, H.; Cao, C.; Ji, W.; Okano, H.; Roberts, R. J.; Ronaghi, M.; Yeung, K.; Zhang, F.; Lee, S. Y. The Power of Synthetic Biology for Bioproduction, Remediation and Pollution Control: The UN’s Sustainable Development Goals Will Inevitably Require the Application of Molecular Biology and Biotechnology on a Global Scale. EMBO Rep 2018,  DOI: 10.15252/embr.201745658
      There is no corresponding record for this reference.
    75. 75
      Thavarajah, W.; Verosloff, M. S.; Jung, J. K.; Alam, K. K.; Miller, J. D.; Jewett, M. C.; Young, S. L.; Lucks, J. B. A Primer on Emerging Field-Deployable Synthetic Biology Tools for Global Water Quality Monitoring. NPJ. Clean Water 2020,  DOI: 10.1038/s41545-020-0064-8
      There is no corresponding record for this reference.
    76. 76
      Gupta, R. M.; Schnitzler, G. R.; Fang, S.; Lee-Kim, V. S.; Barry, A. Multiomic Analysis and CRISPR Perturbation Screens Identify Endothelial Cell Programs and Novel Therapeutic Targets for Coronary Artery Disease. Arterioscler. Thromb. Vasc. Biol. 2023, 43 (5), 600– 608,  DOI: 10.1161/ATVBAHA.123.318328
      There is no corresponding record for this reference.
    77. 77
      Virchow, R. L. K. Disease, Life and Man: Selected Essays; Rather, L. J., Translator; Stanford University Press, 1958.
      There is no corresponding record for this reference.
    78. 78
      Center for Biologics Evaluation; Research. Approved Cellular and Gene Therapy Products. U.S. Food and Drug Administration. https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products (accessed on August 21, 2023).
      There is no corresponding record for this reference.
    79. 79
      Bashor, C. J.; Hilton, I. B.; Bandukwala, H.; Smith, D. M.; Veiseh, O. Engineering the next Generation of Cell-Based Therapeutics. Nat. Rev. Drug Discovery 2022, 21 (9), 655– 675,  DOI: 10.1038/s41573-022-00476-6
      There is no corresponding record for this reference.
    80. 80
      Rosenbaum, L. Tragedy, Perseverance, and Chance - The Story of CAR-T Therapy. N. Engl. J. Med. 2017, 377 (14), 1313– 1315,  DOI: 10.1056/NEJMp1711886
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    81. 81
      Hay, A. E.; Cheung, M. C. CAR T-Cells: Costs, Comparisons, and Commentary. J. Med. Econ. 2019, 22 (7), 613– 615,  DOI: 10.1080/13696998.2019.1582059
      There is no corresponding record for this reference.
    82. 82
      Sampson, K.; Sorenson, C.; Adamala, K. FDA needs to get ready to evaluate synthetic cells, the next generation of therapeutics. STAT. https://www.statnews.com/2022/07/26/fda-develop-framework-evaluate-synthetic-cells/ (accessed on January 6, 2024).
      There is no corresponding record for this reference.
    83. 83
      Lussier, F.; Staufer, O.; Platzman, I.; Spatz, J. P. Can Bottom-Up Synthetic Biology Generate Advanced Drug-Delivery Systems?. Trends Biotechnol. 2021, 39 (5), 445– 459,  DOI: 10.1016/j.tibtech.2020.08.002
      83
      Can Bottom-Up Synthetic Biology Generate Advanced Drug-Delivery Systems
      Lussier, Felix; Staufer, Oskar; Platzman, Ilia; Spatz, Joachim P.
      Trends in Biotechnology (2021), 39 (5), 445-459CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
      A review. Creating a magic bullet that can selectively kill cancer cells while sparing nearby healthy cells remains one of the most ambitious objectives in pharmacol. Nanomedicine, which relies on the use of nanotechnologies to fight disease, was envisaged to fulfill this coveted goal. Despite substantial progress, the structural complexity of therapeutic vehicles impedes their broad clin. application. Novel modular manufg. approaches for engineering programmable drug carriers may be able to overcome some fundamental limitations of nanomedicine. We discuss how bottom-up synthetic biol. principles, empowered by microfluidics, can palliate current drug carrier assembly limitations, and we demonstrate how such a magic bullet could be engineered from the bottom up to ultimately improve clin. outcomes for patients.
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    84. 84
      Staufer, O.; Dietrich, F.; Rimal, R.; Schröter, M.; Fabritz, S.; Boehm, H.; Singh, S.; Möller, M.; Platzman, I.; Spatz, J. P. Bottom-up Assembly of Biomedical Relevant Fully Synthetic Extracellular Vesicles. Sci. Adv. 2021, 7 (36), eabg6666,  DOI: 10.1126/sciadv.abg6666
      There is no corresponding record for this reference.
    85. 85
      Ghaemmaghamian, Z.; Zarghami, R.; Walker, G.; O’Reilly, E.; Ziaee, A. Stabilizing Vaccines via Drying: Quality by Design Considerations. Adv. Drug Delivery Rev. 2022, 187, 114313,  DOI: 10.1016/j.addr.2022.114313
      85
      Stabilizing vaccines via drying: Quality by design considerations
      Ghaemmaghamian, Zahra; Zarghami, Reza; Walker, Gavin; O'Reilly, Emmet; Ziaee, Ahmad
      Advanced Drug Delivery Reviews (2022), 187 (), 114313CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)
      A review. Pandemics and epidemics are continually challenging human beings' health and imposing major stresses on the societies particularly over the last few decades, when their frequency has increased significantly. Protecting humans from multiple diseases is best achieved through vaccination. However, vaccines thermal instability has always been a hurdle in their widespread application, esp. in less developed countries. Furthermore, insufficient vaccine processing capacity is also a major challenge for global vaccination programs. Continuous drying of vaccine formulations is one of the potential solns. to these challenges. This review highlights the challenges on implementing the continuous drying techniques for drying vaccines. The conventional drying methods, emerging technologies and their adaptation by biopharmaceutical industry are investigated considering the patented technologies for drying of vaccines. Moreover, the current progress in applying Quality by Design (QbD) in each of the drying techniques considering the crit. quality attributes (CQAs), crit. process parameters (CPPs) are comprehensively reviewed. An expert advice is presented on the required actions to be taken within the biopharmaceutical industry to move towards continuous stabilization of vaccines in the realm of QbD.
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    86. 86
      Adiga, R.; Al-Adhami, M.; Andar, A.; Borhani, S.; Brown, S.; Burgenson, D.; Cooper, M. A.; Deldari, S.; Frey, D. D.; Ge, X.; Guo, H.; Gurramkonda, C.; Jensen, P.; Kostov, Y.; LaCourse, W.; Liu, Y.; Moreira, A.; Mupparapu, K.; Peñalber-Johnstone, C.; Pilli, M.; Punshon-Smith, B.; Rao, A.; Rao, G.; Rauniyar, P.; Snovida, S.; Taurani, K.; Tilahun, D.; Tolosa, L.; Tolosa, M.; Tran, K.; Vattem, K.; Veeraraghavan, S.; Wagner, B.; Wilhide, J.; Wood, D. W.; Zuber, A. Point-of-Care Production of Therapeutic Proteins of Good-Manufacturing-Practice Quality. Nat. Biomed Eng. 2018, 2 (9), 675– 686,  DOI: 10.1038/s41551-018-0259-1
      86
      Point-of-care production of therapeutic proteins of good-manufacturing-practice quality
      Adiga, Rajani; Al-adhami, Mustafa; Andar, Abhay; Borhani, Shayan; Brown, Sheniqua; Burgenson, David; Cooper, Merideth A.; Deldari, Sevda; Frey, Douglas D.; Ge, Xudong; Guo, Hui; Gurramkonda, Chandrasekhar; Jensen, Penny; Kostov, Yordan; LaCourse, William; Liu, Yang; Moreira, Antonio; Mupparapu, KarunaSri; Penalber-Johnstone, Chariz; Pilli, Manohar; Punshon-Smith, Benjamin; Rao, Aniruddha; Rao, Govind; Rauniyar, Priyanka; Snovida, Sergei; Taurani, Kanika; Tilahun, Dagmawi; Tolosa, Leah; Tolosa, Michael; Tran, Kevin; Vattem, Krishna; Veeraraghavan, Sudha; Wagner, Brandon; Wilhide, Joshua; Wood, David W.; Zuber, Adil
      Nature Biomedical Engineering (2018), 2 (9), 675-686CODEN: NBEAB3; ISSN:2157-846X. (Nature Research)
      Manufg. technologies for biologics rely on large, centralized, good-manufg.-practice (GMP) prodn. facilities and on a cumbersome product-distribution network. Here, we report the development of an automated and portable medicines-on-demand device that enables consistent, small-scale GMP manufg. of therapeutic-grade biologics on a timescale of hours. The device couples the in vitro translation of target proteins from ribosomal DNA, using exts. from reconstituted lyophilized Chinese hamster ovary cells, with the continuous purifn. of the proteins. We used the device to reproducibly manuf. His-tagged granulocyte-colony stimulating factor, erythropoietin, glucose-binding protein and diphtheria toxoid DT5. Medicines-on-demand technol. may enable the rapid manufg. of biologics at the point of care.
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    87. 87
      Ginsburg, G. S.; Phillips, K. A. Precision Medicine: From Science To Value. Health Aff. 2018, 37 (5), 694– 701,  DOI: 10.1377/hlthaff.2017.1624
      There is no corresponding record for this reference.
    88. 88
      Agarwal, S.; Saha, S.; Balla, V. K.; Pal, A.; Barui, A.; Bodhak, S. Current Developments in 3D Bioprinting for Tissue and Organ Regeneration-A Review. Front. Mech. Eng. Chin 2020,  DOI: 10.3389/fmech.2020.589171
      There is no corresponding record for this reference.
    89. 89
      Ghosh, S.; Yi, H.-G. A Review on Bioinks and Their Application in Plant Bioprinting. Int. J. Bioprint 2022, 8 (4), 612,  DOI: 10.18063/ijb.v8i4.612
      There is no corresponding record for this reference.
    90. 90
      Santomartino, R.; Averesch, N. J. H.; Bhuiyan, M.; Cockell, C. S.; Colangelo, J.; Gumulya, Y.; Lehner, B.; Lopez-Ayala, I.; McMahon, S.; Mohanty, A.; Santa Maria, S. R.; Urbaniak, C.; Volger, R.; Yang, J.; Zea, L. Toward Sustainable Space Exploration: A Roadmap for Harnessing the Power of Microorganisms. Nat. Commun. 2023, 14 (1), 1391,  DOI: 10.1038/s41467-023-37070-2
      There is no corresponding record for this reference.
    91. 91
      Rothschild, L. J. Synthetic Biology Meets Bioprinting: Enabling Technologies for Humans on Mars (and Earth). Biochem. Soc. Trans. 2016, 44 (4), 1158– 1164,  DOI: 10.1042/BST20160067
      91
      Synthetic biology meets bioprinting: enabling technologies for humans on Mars (and Earth)
      Rothschild, Lynn J.
      Biochemical Society Transactions (2016), 44 (4), 1158-1164CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
      Human exploration off planet is severely limited by the cost of launching materials into space and by re-supply. Thus materials brought from Earth must be light, stable and reliable at destination. Using traditional approaches, a lunar or Mars base would require either transporting a hefty store of metals or heavy manufg. equipment and construction materials for in situ extn.; both would severely limit any other mission objectives. Long-term human space presence requires periodic replenishment, adding a massive cost overhead. Even robotic missions often sacrifice science goals for heavy radiation and thermal protection. Biol. has the potential to solve these problems because life can replicate and repair itself, and perform a wide variety of chem. reactions including making food, fuel and materials. Synthetic biol. enhances and expands life's evolved repertoire. Using organisms as feedstock, additive manufg. through bioprinting will make possible the dream of producing bespoke tools, food, smart fabrics and even replacement organs on demand. This new approach and the resulting novel products will enable human exploration and settlement on Mars, while providing new manufg. approaches for life on Earth.
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    92. 92
      Averesch, N. J. H.; Berliner, A. J.; Nangle, S. N.; Zezulka, S.; Vengerova, G. L.; Ho, D.; Casale, C. A.; Lehner, B. A. E.; Snyder, J. E.; Clark, K. B.; Dartnell, L. R.; Criddle, C. S.; Arkin, A. P. Microbial Biomanufacturing for Space-Exploration-What to Take and When to Make. Nat. Commun. 2023, 14 (1), 2311,  DOI: 10.1038/s41467-023-37910-1
      There is no corresponding record for this reference.
    93. 93
      Averesch, N. J. H. Choice of Microbial System for in-Situ Resource Utilization on Mars. Front. Astron. Space Sci. 2021,  DOI: 10.3389/fspas.2021.700370
      There is no corresponding record for this reference.
    94. 94
      Cockell, C. S. Bridging the Gap between Microbial Limits and Extremes in Space: Space Microbial Biotechnology in the next 15 Years. Microb. Biotechnol. 2022, 15 (1), 29– 41,  DOI: 10.1111/1751-7915.13927
      There is no corresponding record for this reference.
    95. 95
      Buddingh’, B. C.; van Hest, J. C. M. Artificial Cells: Synthetic Compartments with Life-like Functionality and Adaptivity. Acc. Chem. Res. 2017, 50 (4), 769– 777,  DOI: 10.1021/acs.accounts.6b00512
      95
      Artificial Cells: Synthetic Compartments with Life-like Functionality and Adaptivity
      Buddingh', Bastiaan C.; van Hest, Jan C. M.
      Accounts of Chemical Research (2017), 50 (4), 769-777CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Cells are highly advanced microreactors that form the basis of all life. Their fascinating complexity has inspired scientists to create analogs from synthetic and natural components using a bottom-up approach. The ultimate goal here is to assemble a fully man-made cell that displays functionality and adaptivity as advanced as that found in nature, which will not only provide insight into the fundamental processes in natural cells but also pave the way for new applications of such artificial cells. In this Account, the authors highlight their recent work and that of others on the construction of artificial cells. First, the authors will introduce the key features that characterize a living system; next, the authors will discuss how these have been imitated in artificial cells. First, compartmentalization is crucial to sep. the inner chem. milieu from the external environment. Current state-of-the-art artificial cells comprise subcompartments to mimic the hierarchical architecture of eukaryotic cells and tissue. Furthermore, synthetic gene circuits have been used to encode genetic information that creates complex behavior like pulses or feedback. Addnl., artificial cells have to reproduce to maintain a population. Controlled growth and fission of synthetic compartments have been demonstrated, but the extensive regulation of cell division in nature is still unmatched. Here, the authors also point out important challenges the field needs to overcome to realize its full potential. As artificial cells integrate increasing orders of functionality, maintaining a supporting metab. that can regenerate key metabolites becomes crucial. Furthermore, life does not operate in isolation. Natural cells constantly sense their environment, exchange (chem.) signals, and can move toward a chemoattractant. Here, the authors specifically explore recent efforts to reproduce such adaptivity in artificial cells. For instance, synthetic compartments have been produced that can recruit proteins to the membrane upon an external stimulus or modulate their membrane compn. and permeability to control their interaction with the environment. A next step would be the communication of artificial cells with either bacteria or another artificial cell. Indeed, examples of such primitive chem. signaling are presented. Finally, motility is important for many organisms and has, therefore, also been pursued in synthetic systems. Synthetic compartments that were designed to move in a directed, controlled manner have been assembled, and directed movement toward a chem. attractant is among one of the most life-like directions currently under research. Although the bottom-up construction of an artificial cell that can be truly considered "alive" is still an ambitious goal, the recent work discussed in this Account shows that this is an active field with contributions from diverse disciplines like materials chem. and biochem. Notably, research during the past decade has already provided valuable insights into complex synthetic systems with life-like properties. In the future, artificial cells are thought to contribute to an increased understanding of processes in natural cells and provide opportunities to create smart, autonomous, cell-like materials.
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    96. 96
      Ichihashi, N. What Can We Learn from the Construction of in Vitro Replication Systems?. Ann. N.Y. Acad. Sci. 2019, 1447 (1), 144– 156,  DOI: 10.1111/nyas.14042
      96
      What can we learn from the construction of in vitro replication systems?
      Ichihashi Norikazu
      Annals of the New York Academy of Sciences (2019), 1447 (1), 144-156 ISSN:.
      Replication is a central function of living organisms. Several types of replication systems have been constructed in vitro from various molecules, including peptides, DNA, RNA, and proteins. In this review, I summarize the progress in the construction of replication systems over the past few decades and discuss what we can learn from their construction. I introduce various types of replication systems, supporting the feasibility of the spontaneous appearance of replication early in Earth's history. In the latter part of the review, I focus on parasitic replicators, one of the largest obstacles for sustainable replication. Compartmentalization is discussed as a possible solution.
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    97. 97
      Groaz, A.; Moghimianavval, H.; Tavella, F.; Giessen, T. W.; Vecchiarelli, A. G.; Yang, Q.; Liu, A. P. Engineering Spatiotemporal Organization and Dynamics in Synthetic Cells. WIREs Nanomed. Nanobiotechnol. 2021, 13 (3), e1685,  DOI: 10.1002/wnan.1685
      There is no corresponding record for this reference.
    98. 98
      Gaut, N. J.; Adamala, K. P. Reconstituting Natural Cell Elements in Synthetic Cells. Adv. Biol. 2021, 5 (3), e2000188,  DOI: 10.1002/adbi.202000188
      There is no corresponding record for this reference.
    99. 99
      Stal, L. J. Nitrogen Fixation in Cyanobacteria. In eLS; John Wiley & Sons, Ltd: Chichester, UK, 2015; pp 1– 9.  DOI: 10.1002/9780470015902.a0021159.pub2 .
      There is no corresponding record for this reference.
    100. 100
      Poirier, Y.; Antonenkov, V. D.; Glumoff, T.; Hiltunen, J. K. Peroxisomal β-oxidation─A Metabolic Pathway with Multiple Functions. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2006, 1763 (12), 1413– 1426,  DOI: 10.1016/j.bbamcr.2006.08.034
      100
      Peroxisomal β-oxidation-A metabolic pathway with multiple functions
      Poirier, Yves; Antonenkov, Vasily D.; Glumoff, Tuomo; Hiltunen, J. Kalervo
      Biochimica et Biophysica Acta, Molecular Cell Research (2006), 1763 (12), 1413-1426CODEN: BBAMCO; ISSN:0167-4889. (Elsevier Ltd.)
      A review. Fatty acid degrdn. in most organisms occurs primarily via the β-oxidn. cycle. In mammals, β-oxidn. occurs in both mitochondria and peroxisomes, whereas plants and most fungi harbor the β-oxidn. cycle only in the peroxisomes. Although several of the enzymes participating in this pathway in both organelles are similar, some distinct physiol. roles have been uncovered. Recent advances in the structural elucidation of numerous mammalian and yeast enzymes involved in β-oxidn. have shed light on the basis of the substrate specificity for several of them. Of particular interest is the structural organization and function of the type 1 and 2 multifunctional enzyme (MFE-1 and MFE-2), two enzymes evolutionarily distant yet catalyzing the same overall enzymic reactions but via opposite stereochem. New data on the physiol. roles of the various enzymes participating in β-oxidn. have been gathered through the anal. of knockout mutants in plants, yeast and animals, as well as by the use of polyhydroxyalkanoate synthesis from β-oxidn. intermediates as a tool to study carbon flux through the pathway. In plants, both forward and reverse genetics performed on the model plant Arabidopsis thaliana have revealed novel roles for β-oxidn. in the germination process that is independent of the generation of carbohydrates for growth, as well as in embryo and flower development, and the generation of the phytohormone indole-3-acetic acid and the signal mol. jasmonic acid.
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    101. 101
      Murat, D.; Byrne, M.; Komeili, A. Cell Biology of Prokaryotic Organelles. Cold Spring Harb. Perspect. Biol. 2010, 2 (10), a000422,  DOI: 10.1101/cshperspect.a000422
      There is no corresponding record for this reference.
    102. 102
      Nott, T. J.; Craggs, T. D.; Baldwin, A. J. Membraneless Organelles Can Melt Nucleic Acid Duplexes and Act as Biomolecular Filters. Nat. Chem. 2016, 8 (6), 569– 575,  DOI: 10.1038/nchem.2519
      102
      Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters
      Nott, Timothy J.; Craggs, Timothy D.; Baldwin, Andrew J.
      Nature Chemistry (2016), 8 (6), 569-575CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      Membraneless organelles are cellular compartments made from drops of liq. protein inside a cell. These compartments assemble via the phase sepn. of disordered regions of proteins in response to changes in the cellular environment and the cell cycle. Here we demonstrate that the solvent environment within the interior of these cellular bodies behaves more like an org. solvent than like water. One of the most-stable biol. structures known, the DNA double helix, can be melted once inside the liq. droplet, and simultaneously structures formed from regulatory single-stranded nucleic acids are stabilized. Moreover, proteins are shown to have a wide range of absorption or exclusion from these bodies, and can act as importers for otherwise-excluded nucleic acids, which suggests the existence of a protein-mediated trafficking system. A common strategy in org. chem. is to utilize different solvents to influence the behavior of mols. and reactions. These results reveal that cells have also evolved this capability by exploiting the interiors of membraneless organelles.
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    103. 103
      Lyon, A. S.; Peeples, W. B.; Rosen, M. K. A Framework for Understanding the Functions of Biomolecular Condensates across Scales. Nat. Rev. Mol. Cell Biol. 2021, 22 (3), 215– 235,  DOI: 10.1038/s41580-020-00303-z
      103
      A framework for understanding the functions of biomolecular condensates across scales
      Lyon, Andrew S.; Peeples, William B.; Rosen, Michael K.
      Nature Reviews Molecular Cell Biology (2021), 22 (3), 215-235CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)
      Abstr.: Biomol. condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing mols. that act in processes ranging from RNA metab. to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their phys. properties and regulation arise from mol. constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from mol. (modulating the rates of chem. reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochem. and cellular functions of biomol. condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biol.
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    104. 104
      Protter, D. S. W.; Parker, R. Principles and Properties of Stress Granules. Trends Cell Biol. 2016, 26 (9), 668– 679,  DOI: 10.1016/j.tcb.2016.05.004
      104
      Principles and Properties of Stress Granules
      Protter, David S. W.; Parker, Roy
      Trends in Cell Biology (2016), 26 (9), 668-679CODEN: TCBIEK; ISSN:0962-8924. (Elsevier Ltd.)
      A review. Stress granules are assemblies of untranslating messenger ribonucleoproteins (mRNPs) that form from mRNAs stalled in translation initiation. Stress granules form through interactions between mRNA-binding proteins that link together populations of mRNPs. Interactions promoting stress granule formation include conventional protein-protein interactions as well as interactions involving intrinsically disordered regions (IDRs) of proteins. Assembly and disassembly of stress granules are modulated by various post-translational modifications as well as numerous ATP-dependent RNP or protein remodeling complexes, illustrating that stress granules represent an active liq. wherein energy input maintains their dynamic state. Stress granule formation modulates the stress response, viral infection, and signaling pathways. Persistent or aberrant stress granule formation contributes to neurodegenerative disease and some cancers.
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    105. 105
      Roger, A. J.; Muñoz-Gómez, S. A.; Kamikawa, R. The Origin and Diversification of Mitochondria. Curr. Biol. 2017, 27 (21), R1177– R1192,  DOI: 10.1016/j.cub.2017.09.015
      105
      The Origin and Diversification of Mitochondria
      Roger, Andrew J.; Munoz-Gomez, Sergio A.; Kamikawa, Ryoma
      Current Biology (2017), 27 (21), R1177-R1192CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
      A review. Mitochondria are best known for their role in the generation of ATP by aerobic respiration. Yet, research in the past half century has shown that they perform a much larger suite of functions and that these functions can vary substantially among diverse eukaryotic lineages. Despite this diversity, all mitochondria derive from a common ancestral organelle that originated from the integration of an endosymbiotic alphaproteobacterium into a host cell related to Asgard Archaea. The transition from endosymbiotic bacterium to permanent organelle entailed a massive no. of evolutionary changes including the origins of hundreds of new genes and a protein import system, insertion of membrane transporters, integration of metab. and reprodn., genome redn., endosymbiotic gene transfer, lateral gene transfer and the retargeting of proteins. These changes occurred incrementally as the endosymbiont and the host became integrated. Although many insights into this transition have been gained, controversy persists regarding the nature of the original endosymbiont, its initial interactions with the host and the timing of its integration relative to the origin of other features of eukaryote cells. Since the establishment of the organelle, proteins have been gained, lost, transferred and retargeted as mitochondria have specialized into the spectrum of functional types seen across the eukaryotic tree of life.
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    106. 106
      Mehta, A. P.; Supekova, L.; Chen, J.-H.; Pestonjamasp, K.; Webster, P.; Ko, Y.; Henderson, S. C.; McDermott, G.; Supek, F.; Schultz, P. G. Engineering Yeast Endosymbionts as a Step toward the Evolution of Mitochondria. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (46), 11796– 11801,  DOI: 10.1073/pnas.1813143115
      106
      Engineering yeast endosymbionts as a step toward the evolution of mitochondria
      Mehta, Angad P.; Supekova, Lubica; Chen, Jian-Hua; Pestonjamasp, Kersi; Webster, Paul; Ko, Yeonjin; Henderson, Scott C.; McDermott, Gerry; Supek, Frantisek; Schultz, Peter G.
      Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (46), 11796-11801CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in an archaeal host cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between Escherichia coli and Saccharomyces cerevisiae. An ADP/ATP translocase-expressing E. coli provided ATP to a respiration-deficient cox2 yeast mutant and enabled growth of a yeast-E. coli chimera on a nonfermentable carbon source. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. Expression of several SNARE-like proteins in E. coli was also required, likely to block lysosomal degrdn. of intracellular bacteria. This chimeric system was stable for more than 40 doublings, and GFP-expressing E. coli endosymbionts could be obsd. in the yeast by fluorescence microscopy and X-ray tomog. This readily manipulated system should allow exptl. delineation of host-endosymbiont adaptations that occurred during evolution of the current, highly reduced mitochondrial genome.
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    107. 107
      Headen, D. M.; Aubry, G.; Lu, H.; García, A. J. Microfluidic-Based Generation of Size-Controlled, Biofunctionalized Synthetic Polymer Microgels for Cell Encapsulation. Adv. Mater. 2014, 26 (19), 3003– 3008,  DOI: 10.1002/adma.201304880
      107
      Microfluidic-based generation of size-controlled, biofunctionalized synthetic polymer microgels for cell encapsulation
      Headen, Devon M.; Aubry, Guillaume; Lu, Hang; Garcia, Andres J.
      Advanced Materials (Weinheim, Germany) (2014), 26 (19), 3003-3008CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The high potential of synthetic hydrogel microencapsulation for cell and protein therapeutics has been limited by the lack of synthetic polymer systems with tunable capsule size, cytocompatible crosslinking reactions, rapid crosslinking rates, adequate biomol. permeability, and ease of functionalization with bioactive mols. (e.g., adhesive peptides). Using a synthetic hydrogel system with tunable network and crosslinking characteristics and a microfluidics encapsulation platform, we have created an integrated and robust strategy for microencapsulation of cells in which we can control capsule size and local cellular microenvironment. Addnl., microgel network structure can be tuned to optimize permeability of the capsule to mols. of various sizes. We have demonstrated, proof of concept with two different clin. relevant human cell types, but the versatility of this strategy will allow it to be tailored to fit diverse engineering applications.
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    108. 108
      Torre, P.; Keating, C. D.; Mansy, S. S. Multiphase Water-in-Oil Emulsion Droplets for Cell-Free Transcription-Translation. Langmuir 2014, 30 (20), 5695– 5699,  DOI: 10.1021/la404146g
      108
      Multiphase water-in-oil emulsion droplets for cell-free transcription-translation
      Torre, Paola; Keating, Christine D.; Mansy, Sheref S.
      Langmuir (2014), 30 (20), 5695-5699CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      The construction of genetically encoded cellular mimics in compartments contg. organized synthetic cytosols is desirable for the development of artificial cells. Phase sepd. aq. domains were placed within water-in-oil emulsion droplets in a manner compatible with transcription and translation machinery. Aq. two-phase and three-phase systems (ATPS and A3PS) were assembled with dextran, poly(ethylene glycol), and Ficoll. Aq. two-phase systems were capable of supporting the cell-free expression of protein within water droplets, whereas the aq. three-phase-based system did not give rise to detectable protein synthesis. The expressed protein preferentially partitioned to the dextran-enriched phase. The system could serve as a foundation for building cellular mimics with liq. organelles.
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    109. 109
      Zhang, Y.; Kojima, T.; Kim, G.-A.; McNerney, M. P.; Takayama, S.; Styczynski, M. P. Protocell Arrays for Simultaneous Detection of Diverse Analytes. Nat. Commun. 2021, 12 (1), 5724,  DOI: 10.1038/s41467-021-25989-3
      109
      Protocell arrays for simultaneous detection of diverse analytes
      Zhang, Yan; Kojima, Taisuke; Kim, Ge-Ah; McNerney, Monica P.; Takayama, Shuichi; Styczynski, Mark P.
      Nature Communications (2021), 12 (1), 5724CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Simultaneous detection of multiple analytes from a single sample (multiplexing), particularly when done at the point of need, can guide complex decision-making without increasing the required sample vol. or cost per test. Despite recent advances, multiplexed analyte sensing still typically faces the crit. limitation of measuring only one type of mol. (e.g., small mols. or nucleic acids) per assay platform. Here, we address this bottleneck with a customizable platform that integrates cell-free expression (CFE) with a polymer-based aq. two-phase system (ATPS), producing membrane-less protocells contg. transcription and translation machinery used for detection. We show that multiple protocells, each performing a distinct sensing reaction, can be arrayed in the same microwell to detect chem. diverse targets from the same sample. Furthermore, these protocell arrays are compatible with human biofluids, maintain function after lyophilization and rehydration, and can produce visually interpretable readouts, illustrating this platforms potential as a minimal-equipment, field-deployable, multi-analyte detection tool.
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    110. 110
      Allen, M. E.; Hindley, J. W.; Baxani, D. K.; Ces, O.; Elani, Y. Hydrogels as Functional Components in Artificial Cell Systems. Nat. Rev. Chem. 2022, 6 (8), 562– 578,  DOI: 10.1038/s41570-022-00404-7
      110
      Hydrogels as functional components in artificial cell systems
      Allen, Matthew E.; Hindley, James W.; Baxani, Divesh K.; Ces, Oscar; Elani, Yuval
      Nature Reviews Chemistry (2022), 6 (8), 562-578CODEN: NRCAF7; ISSN:2397-3358. (Nature Portfolio)
      A review. Recent years have seen substantial efforts aimed at constructing artificial cells from various mol. components with the aim of mimicking the processes, behaviors and architectures found in biol. systems. Artificial cell development ultimately aims to produce model constructs that progress our understanding of biol., as well as forming the basis for functional bio-inspired devices that can be used in fields such as therapeutic delivery, biosensing, cell therapy and bioremediation. Typically, artificial cells rely on a bilayer membrane chassis and have fluid aq. interiors to mimic biol. cells. However, a desire to more accurately replicate the gel-like properties of intracellular and extracellular biol. environments has driven increasing efforts to build cell mimics based on hydrogels. This has enabled researchers to exploit some of the unique functional properties of hydrogels that have seen them deployed in fields such as tissue engineering, biomaterials and drug delivery. In this Review, we explore how hydrogels can be leveraged in the context of artificial cell development. We also discuss how hydrogels can potentially be incorporated within the next generation of artificial cells to engineer improved biol. mimics and functional microsystems.
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    111. 111
      Has, C.; Sunthar, P. A Comprehensive Review on Recent Preparation Techniques of Liposomes. J. Liposome Res. 2020, 30 (4), 336– 365,  DOI: 10.1080/08982104.2019.1668010
      111
      A comprehensive review on recent preparation techniques of liposomes
      Has, C.; Sunthar, P.
      Journal of Liposome Research (2020), 30 (4), 336-365CODEN: JLREE7; ISSN:0898-2104. (Taylor & Francis Ltd.)
      A review Liposomes (or lipid vesicles) are a versatile platform as carriers for the delivery of the drugs and other macromols. into human and animal bodies. Though the method of using liposomes has been known since 1960s, and major developments and commercialization of liposomal formulations took place in the late nineties (or early part of this century), newer methods of liposome synthesis and drug encapsulation continue to be an active area of research. With the developments in related fields, such as electrohydrodynamics and microfluidics, and a growing understanding of the mechanisms of lipid assembly from colloidal and intermol. forces, liposome prepn. techniques have been enriched and more predictable than before. This has led to better methods that can also scale at an industrial prodn. level. In this review, we present several novel methods that were introduced over the last decade and compare their advantages over conventional methods. Researchers beginning to explore liposomal formulations will find this resource useful to give an overall direction for an appropriate choice of method. Where possible, we have also provided the known mechanisms behind the prepn. methods.
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    112. 112
      Luisi, P. L.; Allegretti, M.; Pereira de Souza, T.; Steiniger, F.; Fahr, A.; Stano, P. Spontaneous Protein Crowding in Liposomes: A New Vista for the Origin of Cellular Metabolism. Chembiochem 2010, 11 (14), 1989– 1992,  DOI: 10.1002/cbic.201000381
      There is no corresponding record for this reference.
    113. 113
      Michelon, M.; Huang, Y.; de la Torre, L. G.; Weitz, D. A.; Cunha, R. L. Single-Step Microfluidic Production of W/O/W Double Emulsions as Templates for β-Carotene-Loaded Giant Liposomes Formation. Chem. Eng. J. 2019, 366, 27– 32,  DOI: 10.1016/j.cej.2019.02.021
      113
      Single-step microfluidic production of W/O/W double emulsions as templates for β-carotene-loaded giant liposomes formation
      Michelon, Mariano; Huang, Yuting; de la Torre, Lucimara Gaziola; Weitz, David A.; Cunha, Rosiane Lopes
      Chemical Engineering Journal (Amsterdam, Netherlands) (2019), 366 (), 27-32CODEN: CMEJAJ; ISSN:1385-8947. (Elsevier B.V.)
      We demonstrated the microfluidic prodn. of W/O/W double emulsion droplets aiming formation of β-carotene-incorporated giant liposomes for food and/or pharmaceutical applications. For this purpose, glass-capillary microfluidic devices were fabricated to create a truly three-dimensional flow aiming prodn. of giant unilamellar liposomes by solvent evapn. process after W/O/W double emulsion droplet templates formation. A great challenge of microfluidic prodn. of monodisperse and stable W/O/W double emulsion templates for this proposal is the replacement of org. solvents potentially toxic for phospholipids dissoln. Besides, the high cost of several semi-synthetic phospholipids commonly used for giant liposome formation remains as a major technol. challenge to be overcome. Thus, β-carotene-incorporated giant liposomes were generated using biocompatible solvents with low toxic potential (Et acetate and pentane) and non-purified soybean lecithin - a food-grade phospholipid mixt. with low cost - by dewetting and evapn. of the solvents forming the oily intermediate phase of W/O/W double emulsion droplet templates. Our results showed monodisperse β-carotene-loaded giant liposomes with diam. ranging between 100 μm and 180 μm and a stability of approx. 7 days. In this way, a single-step microfluidic process with highly accurate control of size distribution was developed. This microfluidic process proposed is potentially useful for a broad range of applications in protection and delivery of active compds.
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    114. 114
      Elani, Y.; Trantidou, T.; Wylie, D.; Dekker, L.; Polizzi, K.; Law, R. V.; Ces, O. Constructing Vesicle-Based Artificial Cells with Embedded Living Cells as Organelle-like Modules. Sci. Rep. 2018, 8 (1), 4564,  DOI: 10.1038/s41598-018-22263-3
      114
      Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules
      Elani Yuval; Trantidou Tatiana; Wylie Douglas; Law Robert V; Ces Oscar; Elani Yuval; Wylie Douglas; Ces Oscar; Dekker Linda; Polizzi Karen
      Scientific reports (2018), 8 (1), 4564 ISSN:.
      There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.
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    115. 115
      Tayeb, H. H.; Sainsbury, F. Nanoemulsions in Drug Delivery: Formulation to Medical Application. Nanomedicine 2018, 13 (19), 2507– 2525,  DOI: 10.2217/nnm-2018-0088
      115
      Nanoemulsions in drug delivery: formulation to medical application
      Tayeb, Hossam H.; Sainsbury, Frank
      Nanomedicine (London, United Kingdom) (2018), 13 (19), 2507-2525CODEN: NLUKAC; ISSN:1748-6963. (Future Medicine Ltd.)
      Nanoscale oil-in-water emulsions (NEs), heterogeneous systems of two immiscible liqs. stabilized by emulsifiers or surfactants, show great potential in medical applications because of their attractive characteristics for drug delivery. NEs have been explored as therapeutic carriers for hydrophobic compds. via various routes of administration. NEs provide opportunities to improve drug delivery via alternative administration routes. However, deep understanding of the NE manufg. and functionalization fundamentals, and how they relate to the choice of administration route and pharmacol. profile is still needed to ease the clin. translation of NEs. Here, we review the diversity of medical applications for NEs and how that governs their formulation, route of administration, and the emergence of increasing sophistication in NE design for specific application.
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    116. 116
      Vogele, K.; Frank, T.; Gasser, L.; Goetzfried, M. A.; Hackl, M. W.; Sieber, S. A.; Simmel, F. C.; Pirzer, T. Towards Synthetic Cells Using Peptide-Based Reaction Compartments. Nat. Commun. 2018, 9 (1), 3862,  DOI: 10.1038/s41467-018-06379-8
      116
      Towards synthetic cells using peptide-based reaction compartments
      Vogele Kilian; Frank Thomas; Gasser Lukas; Goetzfried Marisa A; Simmel Friedrich C; Pirzer Tobias; Hackl Mathias W; Sieber Stephan A; Simmel Friedrich C
      Nature communications (2018), 9 (1), 3862 ISSN:.
      Membrane compartmentalization and growth are central aspects of living cells, and are thus encoded in every cell's genome. For the creation of artificial cellular systems, genetic information and production of membrane building blocks will need to be coupled in a similar manner. However, natural biochemical reaction networks and membrane building blocks are notoriously difficult to implement in vitro. Here, we utilized amphiphilic elastin-like peptides (ELP) to create self-assembled vesicular structures of about 200 nm diameter. In order to genetically encode the growth of these vesicles, we encapsulate a cell-free transcription-translation system together with the DNA template inside the peptide vesicles. We show in vesiculo production of a functioning fluorescent RNA aptamer and a fluorescent protein. Furthermore, we implement in situ expression of the membrane peptide itself and finally demonstrate autonomous vesicle growth due to the incorporation of this ELP into the membrane.
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    117. 117
      Mushnoori, S.; Lu, C. Y.; Schmidt, K.; Zang, E.; Dutt, M. Peptide-Based Vesicles and Droplets: A Review. J. Phys.: Condens. Matter 2020, 33 (5), 053002,  DOI: 10.1088/1361-648X/abb995
      There is no corresponding record for this reference.
    118. 118
      Sharma, B.; Ma, Y.; Hiraki, H. L.; Baker, B. M.; Ferguson, A. L.; Liu, A. P. Facile Formation of Giant Elastin-like Polypeptide Vesicles as Synthetic Cells. Chem. Commun. 2021, 57 (97), 13202– 13205,  DOI: 10.1039/D1CC05579H
      118
      Facile formation of giant elastin-like polypeptide vesicles as synthetic cells
      Sharma, Bineet; Ma, Yutao; Hiraki, Harrison L.; Baker, Brendon M.; Ferguson, Andrew L.; Liu, Allen P.
      Chemical Communications (Cambridge, United Kingdom) (2021), 57 (97), 13202-13205CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      We demonstrate the facile and robust generation of giant peptide vesicles by using an emulsion transfer method. These robust vesicles can sustain chem. and phys. stresses. The peptide vesicles can host cell-free expression reactions by encapsulating essential ingredients. We show the incorporation of another cell-free expressed elastin-like polypeptide into the existing membrane of the peptide vesicles.
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    119. 119
      Vieregg, J. R.; Lueckheide, M.; Marciel, A. B.; Leon, L.; Bologna, A. J.; Rivera, J. R.; Tirrell, M. V. Oligonucleotide-Peptide Complexes: Phase Control by Hybridization. J. Am. Chem. Soc. 2018, 140 (5), 1632– 1638,  DOI: 10.1021/jacs.7b03567
      119
      Oligonucleotide-peptide complexes: Phase control by hybridization
      Vieregg, Jeffrey R.; Lueckheide, Michael; Marciel, Amanda B.; Leon, Lorraine; Bologna, Alex J.; Rivera, Josean Reyes; Tirrell, Matthew V.
      Journal of the American Chemical Society (2018), 140 (5), 1632-1638CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      When oppositely charged polymers are mixed, counterion release drives phase sepn.; understanding this process is a key unsolved problem in polymer science and biophys. chem., particularly for nucleic acids, polyanions whose biol. functions are intimately related to their high charge d. In the cell, complexation by basic proteins condenses DNA into chromatin, and membraneless organelles formed by liq.-liq. phase sepn. of RNA and proteins perform vital functions and have been linked to disease. Electrostatic interactions are also the primary method used for assembly of nanoparticles to deliver therapeutic nucleic acids into cells. This work describes complexation expts. with oligonucleotides and cationic peptides spanning a wide range of polymer lengths, concns., and structures, including RNA and methylphosphonate backbones. We find that the phase of the complexes is controlled by the hybridization state of the nucleic acid, with double-stranded nucleic acids forming solid ppts. while single-stranded oligonucleotides form liq. coacervates, apparently due to their lower charge d. Adding salt "melts" ppts. into coacervates, and oligonucleotides in coacervates remain competent for sequence-specific hybridization and phase change, suggesting the possibility of environmentally responsive complexes and nanoparticles for therapeutic or sensing applications.
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    120. 120
      Fraccia, T. P.; Jia, T. Z. Liquid Crystal Coacervates Composed of Short Double-Stranded DNA and Cationic Peptides. ACS Nano 2020, 14 (11), 15071– 15082,  DOI: 10.1021/acsnano.0c05083
      120
      Liquid Crystal Coacervates Composed of Short Double-Stranded DNA and Cationic Peptides
      Fraccia, Tommaso P.; Jia, Tony Z.
      ACS Nano (2020), 14 (11), 15071-15082CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Phase sepn. of nucleic acids and proteins is a ubiquitous phenomenon regulating subcellular compartment structure and function. While complex coacervation of flexible single-stranded nucleic acids is broadly investigated, coacervation of double-stranded DNA (dsDNA) is less studied because of its propensity to generate solid ppts. Here, we reverse this perspective by showing that short dsDNA and poly-L-lysine coacervates can escape pptn. while displaying a surprisingly complex phase diagram, including the full set of liq. crystal (LC) mesophases obsd. to date in bulk dsDNA. Short dsDNA supramol. aggregation and packing in the dense coacervate phase are the main parameters regulating the global LC-coacervate phase behavior. LC-coacervate structure was characterized upon variations in temp. and monovalent salt, DNA, and peptide concns., which allow continuous reversible transitions between all accessible phases. A deeper understanding of LC-coacervates can gain insights to decipher structures and phase transition mechanisms within biomol. condensates, to design stimuli-responsive multiphase synthetic compartments with different degrees of order and to exploit self-assembly driven cooperative prebiotic evolution of nucleic acids and peptides.
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    121. 121
      Aumiller, W. M., Jr; Pir Cakmak, F.; Davis, B. W.; Keating, C. D. RNA-Based Coacervates as a Model for Membraneless Organelles: Formation, Properties, and Interfacial Liposome Assembly. Langmuir 2016, 32 (39), 10042– 10053,  DOI: 10.1021/acs.langmuir.6b02499
      121
      RNA-Based Coacervates as a Model for Membraneless Organelles: Formation, Properties, and Interfacial Liposome Assembly
      Aumiller, William M.; Pir Cakmak, Fatma; Davis, Bradley W.; Keating, Christine D.
      Langmuir (2016), 32 (39), 10042-10053CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Liq.-liq. phase sepn. is responsible for the formation of P granules, nucleoli, and other membraneless subcellular organelles composed of RNA and proteins. Efforts to understand the phys. basis of liq. organelle formation have thus far focused on intrinsically disordered proteins (IDPs) as major components that dictate occurrence and properties. Here, the authors show that complex coacervates composed of low complexity RNA [polyuridylic acid, poly(U)] and short polyamines (spermine and spermidine) share many features of IDP-based coacervates. Poly(U)/polyamine coacervates compartmentalized biomols. (peptides, oligonucleotides) in a sequence- and length- dependent manner. These solutes retained mobility within the coacervate droplets, as demonstrated by rapid recovery from photobleaching. Coacervation was reversible with changes in soln. temp. due to changes in the poly(U) structure that impacted its interactions with polyamines. The authors further demonstrated that lipid vesicles assembled at the droplet interface without impeding RNA entry/egress. These vesicles remained intact at the interface and could be released upon temp.-induced droplet dissoln.
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    122. 122
      Rimoli, M. G.; Rabaioli, M. R.; Melisi, D.; Curcio, A.; Mondello, S.; Mirabelli, R.; Abignente, E. Synthetic Zeolites as a New Tool for Drug Delivery. J. Biomed. Mater. Res., Part A 2008, 87 (1), 156– 164,  DOI: 10.1002/jbm.a.31763
      122
      Synthetic zeolites as a new tool for drug delivery
      Rimoli, Maria G.; Rabaioli, Maria R.; Melisi, Daniela; Curcio, Annalisa; Mondello, Sandro; Mirabelli, Rosella; Abignente, Enrico
      Journal of Biomedical Materials Research, Part A (2008), 87A (1), 156-164CODEN: JBMRCH; ISSN:1549-3296. (John Wiley & Sons, Inc.)
      Synthetic zeolites were studied in order to investigate their ability to encapsulate and to release drugs. In particular, a zeolite X and a zeolitic product obtained from a cocrystn. of zeolite X and zeolite A were examd. These materials were characterized by chem. analyses (ICP-AES), x-ray diffraction, nitrogen adsorption isotherm, SEM, laser diffraction, and IR spectroscopy. Since ketoprofen was chosen as a model drug for the formulation of controlled-release dosage forms, it was encapsulated into these two types of synthetic zeolites by a soaking procedure. Drug-loaded matrixes were then characterized for entrapped drug amt. and thermogravimetric behavior. In both types of activated zeolites, the total amt. of ketoprofen (800 mg) was encapsulated in 2 g of matrix. By using HPLC measurements, ketoprofen release studies were done at different pH conditions so as to mimic gastrointestinal fluids. The absence of release in acid conditions and a double phased release, at two different pH values (5 and 6.8), suggest that after activation these materials offer good potential for a modified release delivery system of ketoprofen.
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    123. 123
      Hyman, A. A.; Weber, C. A.; Jülicher, F. Liquid-Liquid Phase Separation in Biology. Annu. Rev. Cell Dev. Biol. 2014, 30, 39– 58,  DOI: 10.1146/annurev-cellbio-100913-013325
      123
      Liquid-liquid phase separation in biology
      Hyman, Anthony A.; Weber, Christoph A.; Juelicher, Frank
      Annual Review of Cell and Developmental Biology (2014), 30 (), 39-58CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews)
      A review. Cells organize many of their biochem. reactions in non-membrane compartments. Recent evidence showed that many of these compartments are liqs. that form by phase sepn. from the cytoplasm. Here the basic phys. concepts necessary to understand the consequences of liq.-like states for biol. functions are discussed.
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    124. 124
      Junge, F.; Haberstock, S.; Roos, C.; Stefer, S.; Proverbio, D.; Dötsch, V.; Bernhard, F. Advances in Cell-Free Protein Synthesis for the Functional and Structural Analysis of Membrane Proteins. N. Biotechnol. 2011, 28 (3), 262– 271,  DOI: 10.1016/j.nbt.2010.07.002
      There is no corresponding record for this reference.
    125. 125
      Lee, K. Y.; Park, S.-J.; Lee, K. A.; Kim, S.-H.; Kim, H.; Meroz, Y.; Mahadevan, L.; Jung, K.-H.; Ahn, T. K.; Parker, K. K.; Shin, K. Photosynthetic Artificial Organelles Sustain and Control ATP-Dependent Reactions in a Protocellular System. Nat. Biotechnol. 2018, 36 (6), 530– 535,  DOI: 10.1038/nbt.4140
      125
      Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system
      Lee, Keel Yong; Park, Sung-Jin; Lee, Keon Ah; Kim, Se-Hwan; Kim, Heeyeon; Meroz, Yasmine; Mahadevan, L.; Jung, Kwang-Hwan; Ahn, Tae Kyu; Parker, Kevin Kit; Shin, Kwanwoo
      Nature Biotechnology (2018), 36 (6), 530-535CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
      Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymn., with the latter altering outer vesicle morphol. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.
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    126. 126
      Jewett, M. C.; Swartz, J. R. Mimicking the Escherichia Coli Cytoplasmic Environment Activates Long-Lived and Efficient Cell-Free Protein Synthesis. Biotechnol. Bioeng. 2004, 86 (1), 19– 26,  DOI: 10.1002/bit.20026
      126
      Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis
      Jewett, Michael C.; Swartz, James R.
      Biotechnology and Bioengineering (2004), 86 (1), 19-26CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
      Cell-free translation systems generally utilize high-energy phosphate compds. to regenerate the ATP necessary to drive protein synthesis. This hampers the widespread use and practical implementation of this technol. in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change. To address these problems, a cell-free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein. The "Cytomim" system, synthesizes chloramphenicol acetyl-transferase (CAT) for up to 6 h in a batch reaction to yield 700 μg/mL of protein. By more closely replicating the physiol. conditions of the cytoplasm of Escherichia coli, the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addn., or the need for expensive high-energy phosphate compds.
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    127. 127
      Caschera, F.; Noireaux, V. Synthesis of 2.3 Mg/mL of Protein with an All Escherichia Coli Cell-Free Transcription-Translation System. Biochimie 2014, 99, 162– 168,  DOI: 10.1016/j.biochi.2013.11.025
      127
      Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system
      Caschera, Filippo; Noireaux, Vincent
      Biochimie (2014), 99 (), 162-168CODEN: BICMBE; ISSN:0300-9084. (Elsevier Masson SAS)
      Cell-free protein synthesis is becoming a useful technique for synthetic biol. As more applications are developed, the demand for novel and more powerful in vitro expression systems is increasing. In this work, an all Escherichia coli cell-free system, that uses the endogenous transcription and translation mol. machineries, is optimized to synthesize up to 2.3 mg/mL of a reporter protein in batch mode reactions. A new metab. based on maltose allows recycling of inorg. phosphate through its incorporation into newly available glucose mols., which are processed through the glycolytic pathway to produce more ATP. As a result, the ATP regeneration is more efficient and cell-free protein synthesis lasts up to 10 h. Using a com. E. coli strain, we show for the first time that more than 2 mg/mL of protein can be synthesized in run-off cell-free transcription-translation reactions by optimizing the energy regeneration and waste products recycling. This work suggests that endogenous enzymes present in the cytoplasmic ext. can be used to implement new metabolic pathways for increasing protein yields. This system is the new basis of a cell-free gene expression platform used to construct and to characterize complex biochem. processes in vitro such as gene circuits.
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    128. 128
      Garamella, J.; Marshall, R.; Rustad, M.; Noireaux, V. The All E. Coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology. ACS Synth. Biol. 2016, 5 (4), 344– 355,  DOI: 10.1021/acssynbio.5b00296
      128
      The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology
      Garamella, Jonathan; Marshall, Ryan; Rustad, Mark; Noireaux, Vincent
      ACS Synthetic Biology (2016), 5 (4), 344-355CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic ext., thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metab. for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on av., the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. MRNAs and protein degrdn., resp. tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biol. systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.
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    129. 129
      Kim, D.-M.; Swartz, J. R. Efficient Production of a Bioactive, Multiple Disulfide-Bonded Protein Using Modified Extracts of Escherichia Coli. Biotechnol. Bioeng. 2004, 85 (2), 122– 129,  DOI: 10.1002/bit.10865
      There is no corresponding record for this reference.
    130. 130
      Jeong, S.; Nguyen, H. T.; Kim, C. H.; Ly, M. N.; Shin, K. Toward Artificial Cells: Novel Advances in Energy Conversion and Cellular Motility. Adv. Funct. Mater. 2020, 30 (11), 1907182,  DOI: 10.1002/adfm.201907182
      130
      Toward Artificial Cells: Novel Advances in Energy Conversion and Cellular Motility
      Jeong, Sungwoo; Nguyen, Huong Thanh; Kim, Chang Ho; Ly, Mai Nguyet; Shin, Kwanwoo
      Advanced Functional Materials (2020), 30 (11), 1907182CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. The demand to discover every single cellular component has been continuously increasing along with the development of biol. techniques. The bottom-up approach to construct a cell-mimicking system from well-defined and tunable compns. is accelerating, with the ultimate goal of comprehending a biol. cell. From among the available techniques, the artificial cell has been increasingly recognized as one of the most powerful tools for building a cell-like system from scratch. This review summarizes the development of artificial cells, from a pure giant unilamellar vesicle (GUV) to a controllable, self-fueled proteoliposome, both of which are highly compartmentalized. The basic components of an artificial cell, as well as the optimal conditions required for successful, reproducible GUV formation and protein reconstitution, are discussed. Most importantly, progress in studying the metabolic reactions in and the motility of a reconstituted artificial cell are the main focus of the review. The ability to perform a complicated reaction cascade in a controllable manner is highlighted as a promising perspective to obtaining an autonomous and movable GUV.
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    131. 131
      Hanczyc, M. M.; Fujikawa, S. M.; Szostak, J. W. Experimental Models of Primitive Cellular Compartments: Encapsulation, Growth, and Division. Science 2003, 302 (5645), 618– 622,  DOI: 10.1126/science.1089904
      131
      Experimental Models of Primitive Cellular Compartments: Encapsulation, Growth, and Division
      Hanczyc, Martin M.; Fujikawa, Shelly M.; Szostak, Jack W.
      Science (Washington, DC, United States) (2003), 302 (5645), 618-622CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The clay montmorillonite is known to catalyze the polymn. of RNA from activated ribonucleotides. Here we report that montmorillonite accelerates the spontaneous conversion of fatty acid micelles into vesicles. Clay particles often become encapsulated in these vesicles, thus providing a pathway for the prebiotic encapsulation of catalytically active surfaces within membrane vesicles. In addn., RNA adsorbed to clay can be encapsulated within vesicles. Once formed, such vesicles can grow by incorporating fatty acid supplied as micelles and can divide without diln. of their contents by extrusion through small pores. These processes mediate vesicle replication through cycles of growth and division. The formation, growth, and division of the earliest cells may have occurred in response to similar interactions with mineral particles and inputs of material and energy.
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    132. 132
      Kretschmer, S.; Ganzinger, K. A.; Franquelim, H. G.; Schwille, P. Synthetic Cell Division via Membrane-Transforming Molecular Assemblies. BMC Biol. 2019, 17 (1), 43,  DOI: 10.1186/s12915-019-0665-1
      132
      Synthetic cell division via membrane-transforming molecular assemblies
      Kretschmer Simon; Ganzinger Kristina A; Franquelim Henri G; Schwille Petra; Kretschmer Simon; Ganzinger Kristina A
      BMC biology (2019), 17 (1), 43 ISSN:.
      Reproduction, i.e. the ability to produce new individuals from a parent organism, is a hallmark of living matter. Even the simplest forms of reproduction require cell division: attempts to create a designer cell therefore should include a synthetic cell division machinery. In this review, we will illustrate how nature solves this task, describing membrane remodelling processes in general and focusing on bacterial cell division in particular. We discuss recent progress made in their in vitro reconstitution, identify open challenges, and suggest how purely synthetic building blocks could provide an additional and attractive route to creating artificial cell division machineries.
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    133. 133
      Anzai, K.; Yoshida, M.; Kirino, Y. Change in Intravesicular Volume of Liposomes by Freeze-Thaw Treatment as Studied by the ESR Stopped-Flow Technique. Biochimica et Biophysica Acta (BBA) - Biomembranes 1990, 1021 (1), 21– 26,  DOI: 10.1016/0005-2736(90)90378-2
      There is no corresponding record for this reference.
    134. 134
      van der Valk, T.; Pečnerová, P.; Díez-Del-Molino, D.; Bergström, A.; Oppenheimer, J.; Hartmann, S.; Xenikoudakis, G.; Thomas, J. A.; Dehasque, M.; Sağlıcan, E.; Fidan, F. R.; Barnes, I.; Liu, S.; Somel, M.; Heintzman, P. D.; Nikolskiy, P.; Shapiro, B.; Skoglund, P.; Hofreiter, M.; Lister, A. M.; Götherström, A.; Dalén, L. Million-Year-Old DNA Sheds Light on the Genomic History of Mammoths. Nature 2021, 591 (7849), 265– 269,  DOI: 10.1038/s41586-021-03224-9
      134
      Million-year-old DNA sheds light on the genomic history of mammoths
      van der Valk, Tom; Pecnerova, Patricia; Diez-del-Molino, David; Bergstroem, Anders; Oppenheimer, Jonas; Hartmann, Stefanie; Xenikoudakis, Georgios; Thomas, Jessica A.; Dehasque, Marianne; Saglican, Ekin; Fidan, Fatma Rabia; Barnes, Ian; Liu, Shanlin; Somel, Mehmet; Heintzman, Peter D.; Nikolskiy, Pavel; Shapiro, Beth; Skoglund, Pontus; Hofreiter, Michael; Lister, Adrian M.; Goetherstroem, Anders; Dalen, Love
      Nature (London, United Kingdom) (2021), 591 (7849), 265-269CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Temporal genomic data hold great potential for studying evolutionary processes such as speciation. However, sampling across speciation events would, in many cases, require genomic time series that stretch well back into the Early Pleistocene subepoch. Although theor. models suggest that DNA should survive on this timescale1, the oldest genomic data recovered so far are from a horse specimen dated to 780-560 thousand years ago2. Here we report the recovery of genome-wide data from three mammoth specimens dating to the Early and Middle Pleistocene subepochs, two of which are more than one million years old. We find that two distinct mammoth lineages were present in eastern Siberia during the Early Pleistocene. One of these lineages gave rise to the woolly mammoth and the other represents a previously unrecognized lineage that was ancestral to the first mammoths to colonize North America. Our analyses reveal that the Columbian mammoth of North America traces its ancestry to a Middle Pleistocene hybridization between these two lineages, with roughly equal admixt. proportions. Finally, we show that the majority of protein-coding changes assocd. with cold adaptation in woolly mammoths were already present one million years ago. These findings highlight the potential of deep-time palaeogenomics to expand our understanding of speciation and long-term adaptive evolution.
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    135. 135
      Li, Z.; Deutscher, M. P. Analyzing the Decay of Stable RNAs in E. Coli. Methods Enzymol. 2008, 447, 31– 45,  DOI: 10.1016/S0076-6879(08)02202-7
      There is no corresponding record for this reference.
    136. 136
      Chan, L. Y.; Mugler, C. F.; Heinrich, S.; Vallotton, P.; Weis, K. Non-Invasive Measurement of mRNA Decay Reveals Translation Initiation as the Major Determinant of mRNA Stability. eLife 2018,  DOI: 10.7554/eLife.32536
      There is no corresponding record for this reference.
    137. 137
      Paul, N.; Joyce, G. F. A Self-Replicating Ligase Ribozyme. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (20), 12733– 12740,  DOI: 10.1073/pnas.202471099
      There is no corresponding record for this reference.
    138. 138
      Robertson, M. P.; Joyce, G. F. Highly Efficient Self-Replicating RNA Enzymes. Chem. Biol. 2014, 21 (2), 238– 245,  DOI: 10.1016/j.chembiol.2013.12.004
      138
      Highly Efficient Self-Replicating RNA Enzymes
      Robertson, Michael P.; Joyce, Gerald F.
      Chemistry & Biology (Oxford, United Kingdom) (2014), 21 (2), 238-245CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
      An RNA enzyme has been developed that catalyzes the joining of oligonucleotide substrates to form addnl. copies of itself, undergoing self-replication with exponential growth. The enzyme also can cross-replicate with a partner enzyme, resulting in their mutual exponential growth and enabling self-sustained Darwinian evolution. The opportunity for inventive evolution within this synthetic genetic system depends on the diversity of the evolving population, which is limited by the catalytic efficiency of the enzyme. Directed evolution was used to improve the efficiency of the enzyme and increase its exponential growth rate to 0.14 min-1, corresponding to a doubling time of 5 min. This is close to the limit of 0.21 min-1 imposed by the rate of product release, but sufficient to enable more than 80 logs of growth per day.
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    139. 139
      Chen, I. A.; Salehi-Ashtiani, K.; Szostak, J. W. RNA Catalysis in Model Protocell Vesicles. J. Am. Chem. Soc. 2005, 127 (38), 13213– 13219,  DOI: 10.1021/ja051784p
      139
      RNA Catalysis in Model Protocell Vesicles
      Chen, Irene A.; Salehi-Ashtiani, Kourosh; Szostak, Jack W.
      Journal of the American Chemical Society (2005), 127 (38), 13213-13219CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We are engaged in a long-term effort to synthesize chem. systems capable of Darwinian evolution, based on the encapsulation of self-replicating nucleic acids in self-replicating membrane vesicles. Here, we address the issue of the compatibility of these two replicating systems. Fatty acids form vesicles that are able to grow and divide, but vesicles composed solely of fatty acids are incompatible with the folding and activity of most ribozymes, because low concns. of divalent cations (e.g., Mg2+) cause fatty acids to ppt. Furthermore, vesicles that grow and divide must be permeable to the cations and substrates required for internal metab. We used a mixt. of myristoleic acid and its glycerol monoester to construct vesicles that were Mg2+-tolerant and found that Mg2+ cations can permeate the membrane and equilibrate within a few minutes. In vesicles encapsulating a hammerhead ribozyme, the addn. of external Mg2+ led to the activation and self-cleavage of the ribozyme mols. Vesicles composed of these amphiphiles grew spontaneously through osmotically driven competition between vesicles, and further modification of the membrane compn. allowed growth following mixed micelle addn. Our results show that membranes made from simple amphiphiles can form vesicles that are stable enough to retain encapsulated RNAs in the presence of divalent cations, yet dynamic enough to grow spontaneously and allow the passage of Mg2+ and mononucleotides without specific macromol. transporters. This combination of stability and dynamics is crit. for building model protocells in the lab. and may have been important for early cellular evolution.
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    140. 140
      Gorbalenya, A. E.; Enjuanes, L.; Ziebuhr, J.; Snijder, E. J. Nidovirales: Evolving the Largest RNA Virus Genome. Virus Res. 2006, 117 (1), 17– 37,  DOI: 10.1016/j.virusres.2006.01.017
      There is no corresponding record for this reference.
    141. 141
      Joyce, G. F.; Szostak, J. W. Protocells and RNA Self-Replication. Cold Spring Harb. Perspect. Biol. 2018, 10 (9), a034801,  DOI: 10.1101/cshperspect.a034801
      141
      Protocells and RNA self-replication
      Joyce, Gerald F.; Szostak, Jack W.
      Cold Spring Harbor Perspectives in Biology (2018), 10 (9), a034801/1-a034801/20CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
      The general notion of an "RNA world" is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA, and RNA mols. were the chief agents of catalytic function. Assuming that all of the components of RNA were available in some prebiotic locale, these components could have assembled into activated nucleotides that condensed to form RNA polymers, setting the stage for the chem. replication of polynucleotides through RNA-templated RNA polymn. If a sufficient diversity of RNAs could be copied with reasonable rate and fidelity, then Darwinian evolution would begin with RNAs that facilitated their own reprodn. enjoying a selective advantage. The concept of a "protocell" refers to a compartment where replication of the primitive genetic material took place and where primitive catalysts gave rise to products that accumulated locally for the benefit of the replicating cellular entity. Replication of both the protocell and its encapsulated genetic material would have enabled natural selection to operate based on the differential fitness of competing cellular entities, ultimately giving rise to modern cellular life.
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    142. 142
      Jahn, M.; Vorpahl, C.; Hübschmann, T.; Harms, H.; Müller, S. Copy Number Variability of Expression Plasmids Determined by Cell Sorting and Droplet Digital PCR. Microb. Cell Fact. 2016, 15 (1), 211,  DOI: 10.1186/s12934-016-0610-8
      There is no corresponding record for this reference.
    143. 143
      Nielsen, A. A. K.; Der, B. S.; Shin, J.; Vaidyanathan, P.; Paralanov, V.; Strychalski, E. A.; Ross, D.; Densmore, D.; Voigt, C. A. Genetic Circuit Design Automation. Science 2016, 352 (6281), aac7341,  DOI: 10.1126/science.aac7341
      There is no corresponding record for this reference.
    144. 144
      Salis, H. M.; Mirsky, E. A.; Voigt, C. A. Automated Design of Synthetic Ribosome Binding Sites to Control Protein Expression. Nat. Biotechnol. 2009, 27 (10), 946– 950,  DOI: 10.1038/nbt.1568
      144
      Automated design of synthetic ribosome binding sites to control protein expression
      Salis, Howard M.; Mirsky, Ethan A.; Voigt, Christopher A.
      Nature Biotechnology (2009), 27 (10), 946-950CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      Microbial engineering often requires fine control over protein expression-for example, to connect genetic circuits or control flux through a metabolic pathway. To circumvent the need for trial and error optimization, we developed a predictive method for designing synthetic ribosome binding sites, enabling a rational control over the protein expression level. Exptl. validation of >100 predictions in Escherichia coli showed that the method is accurate to within a factor of 2.3 over a range of 100,000-fold. The design method also correctly predicted that reusing identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels. We demonstrate the method's utility by rationally optimizing protein expression to connect a genetic sensor to a synthetic circuit. The proposed forward engineering approach should accelerate the construction and systematic optimization of large genetic systems.
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    145. 145
      Ostrov, N.; Beal, J.; Ellis, T.; Gordon, D. B.; Karas, B. J.; Lee, H. H.; Lenaghan, S. C.; Schloss, J. A.; Stracquadanio, G.; Trefzer, A.; Bader, J. S.; Church, G. M.; Coelho, C. M.; Efcavitch, J. W.; Güell, M.; Mitchell, L. A.; Nielsen, A. A. K.; Peck, B.; Smith, A. C.; Stewart, C. N., Jr; Tekotte, H. Technological Challenges and Milestones for Writing Genomes. Science 2019, 366 (6463), 310– 312,  DOI: 10.1126/science.aay0339
      145
      Technological challenges and milestones for writing genomes
      Ostrov, Nili; Beal, Jacob; Ellis, Tom; Gordon, D. Benjamin; Karas, Bogumil J.; Lee, Henry H.; Lenaghan, Scott C.; Schloss, Jeffery A.; Stracquadanio, Giovanni; Trefzer, Axel; Bader, Joel S.; Church, George M.; Coelho, Cintia M.; Efcavitch, J. William; Guell, Marc; Mitchell, Leslie A.; Nielsen, Alec A. K.; Peck, Bill; Smith, Alexander C.; Stewart, C. Neal, Jr.; Tekotte, Hille
      Science (Washington, DC, United States) (2019), 366 (6463), 310-312CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      A review. Engineering biol. with recombinant DNA, broadly called synthetic biol., has progressed tremendously in the last decade, owing to continued industrialization of DNA synthesis, discovery and development of mol. tools and organisms, and increasingly sophisticated modeling and analytic tools. However, we have yet to understand the full potential of engineering biol. because of our inability to write and test whole genomes, which we call synthetic genomics. Substantial improvements are needed to reduce the cost and increase the speed and reliability of genetic tools. Here, we identify emerging technologies and improvements to existing methods that will be needed in four major areas to advance synthetic genomics within the next 10 years: genome design, DNA synthesis, genome editing, and chromosome construction (see table). Similar to other large-scale projects for responsible advancement of innovative technologies, such as the Human Genome Project, an international, cross-disciplinary effort consisting of public and private entities will likely yield maximal return on investment and open new avenues of research and biotechnol.
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    146. 146
      Farasat, I.; Kushwaha, M.; Collens, J.; Easterbrook, M.; Guido, M.; Salis, H. M. Efficient Search, Mapping, and Optimization of Multi-Protein Genetic Systems in Diverse Bacteria. Mol. Syst. Biol. 2014, 10 (6), 731,  DOI: 10.15252/msb.20134955
      146
      Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria
      Farasat Iman; Easterbrook Michael; Guido Matthew; Kushwaha Manish; Collens Jason; Salis Howard M
      Molecular systems biology (2014), 10 (), 731 ISSN:.
      Developing predictive models of multi-protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi-protein expression space across a > 10,000-fold range with tailored search parameters and well-predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram-positive and gram-negative bacterial hosts. We then combined the search algorithm with system-level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence-expression-activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate-limiting steps in metabolism. Creating sequence-expression-activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs.
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    147. 147
      Pretorius, I. S.; Boeke, J. D. Yeast 2.0-Connecting the Dots in the Construction of the World’s First Functional Synthetic Eukaryotic Genome. FEMS Yeast Res. 2018,  DOI: 10.1093/femsyr/foy032
      There is no corresponding record for this reference.
    148. 148
      Blight, K. J.; Kolykhalov, A. A.; Rice, C. M. Efficient Initiation of HCV RNA Replication in Cell Culture. Science 2000, 290 (5498), 1972– 1974,  DOI: 10.1126/science.290.5498.1972
      148
      Efficient initiation of HCV RNA replication in cell culture
      Blight, Keril J.; Kolykhalov, Alexander A.; Rice, Charles M.
      Science (Washington, D. C.) (2000), 290 (5498), 1972-1974CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Hepatitis C virus (HCV) infection is a global health problem affecting an estd. 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity detg. region (ISDR). Independent of the ISDR, IFN-α rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.
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    149. 149
      Gibson, D. G.; Young, L.; Chuang, R.-Y.; Venter, J. C.; Hutchison, C. A., 3rd; Smith, H. O. Enzymatic Assembly of DNA Molecules up to Several Hundred Kilobases. Nat. Methods 2009, 6 (5), 343– 345,  DOI: 10.1038/nmeth.1318
      149
      Enzymatic assembly of DNA molecules up to several hundred kilobases
      Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan; Venter, J. Craig; Hutchison, Clyde A.; Smith, Hamilton O.
      Nature Methods (2009), 6 (5), 343-345CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      The authors describe an isothermal, single-reaction method for assembling multiple overlapping DNA mols. by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First they recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful mol. engineering tool.
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    150. 150
      Venter, J. C.; Glass, J. I.; Hutchison, C. A., 3rd; Vashee, S. Synthetic Chromosomes, Genomes, Viruses, and Cells. Cell 2022, 185 (15), 2708– 2724,  DOI: 10.1016/j.cell.2022.06.046
      150
      Synthetic chromosomes, genomes, viruses, and cells
      Venter, J. Craig; Glass, John I.; Hutchison, Clyde A. III; Vashee, Sanjay
      Cell (Cambridge, MA, United States) (2022), 185 (15), 2708-2724CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      A review. Synthetic genomics is the construction of viruses, bacteria, and eukaryotic cells with synthetic genomes. It involves two basic processes: synthesis of complete genomes or chromosomes and booting up of those synthetic nucleic acids to make viruses or living cells. The first synthetic genomics efforts resulted in the construction of viruses. This led to a revolution in viral reverse genetics and improvements in vaccine design and manuf. The first bacterium with a synthetic genome led to construction of a minimal bacterial cell and recoded Escherichia coli strains able to incorporate multiple non-std. amino acids in proteins and resistant to phage infection. Further advances led to a yeast strain with a synthetic genome and new approaches for animal and plant artificial chromosomes. On the horizon there are dramatic advances in DNA synthesis that will enable extraordinary new opportunities in medicine, industry, agriculture, and research.
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    151. 151
      Fredens, J.; Wang, K.; de la Torre, D.; Funke, L. F. H.; Robertson, W. E.; Christova, Y.; Chia, T.; Schmied, W. H.; Dunkelmann, D. L.; Beránek, V.; Uttamapinant, C.; Llamazares, A. G.; Elliott, T. S.; Chin, J. W. Total Synthesis of Escherichia Coli with a Recoded Genome. Nature 2019, 569 (7757), 514– 518,  DOI: 10.1038/s41586-019-1192-5
      151
      Total synthesis of Escherichia coli with a recoded genome
      Fredens, Julius; Wang, Kaihang; de la Torre, Daniel; Funke, Louise F. H.; Robertson, Wesley E.; Christova, Yonka; Chia, Tiongsun; Schmied, Wolfgang H.; Dunkelmann, Daniel L.; Beranek, Vaclav; Uttamapinant, Chayasith; Llamazares, Andres Gonzalez; Elliott, Thomas S.; Chin, Jason W.
      Nature (London, United Kingdom) (2019), 569 (7757), 514-518CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the no. of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme-with simple corrections at just seven positions-to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential tRNA.
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    152. 152
      Zhao, Y.; Coelho, C.; Hughes, A. L.; Lazar-Stefanita, L.; Yang, S.; Brooks, A. N.; Walker, R. S. K.; Zhang, W.; Lauer, S.; Hernandez, C.; Cai, J.; Mitchell, L. A.; Agmon, N.; Shen, Y.; Sall, J.; Fanfani, V.; Jalan, A.; Rivera, J.; Liang, F.-X.; Bader, J. S.; Stracquadanio, G.; Steinmetz, L. M.; Cai, Y.; Boeke, J. D. Debugging and Consolidating Multiple Synthetic Chromosomes Reveals Combinatorial Genetic Interactions. Cell 2023, 186 (24), 5220– 5236,  DOI: 10.1016/j.cell.2023.09.025
      There is no corresponding record for this reference.
    153. 153
      Woese, C. The Universal Ancestor. Proc. Natl. Acad. Sci. U. S. A. 1998, 95 (12), 6854– 6859,  DOI: 10.1073/pnas.95.12.6854
      There is no corresponding record for this reference.
    154. 154
      Woese, C. R. On the Evolution of Cells. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (13), 8742– 8747,  DOI: 10.1073/pnas.132266999
      There is no corresponding record for this reference.
    155. 155
      Sandberg, T. E.; Wise, K. S.; Dalldorf, C.; Szubin, R.; Feist, A. M.; Glass, J. I.; Palsson, B. O. Adaptive Evolution of a Minimal Organism with a Synthetic Genome. iScience 2023, 26 (9), 107500,  DOI: 10.1016/j.isci.2023.107500
      There is no corresponding record for this reference.
    156. 156
      Moger-Reischer, R. Z.; Glass, J. I.; Wise, K. S.; Sun, L.; Bittencourt, D. M. C.; Lehmkuhl, B. K.; Schoolmaster, D. R., Jr; Lynch, M.; Lennon, J. T. Evolution of a Minimal Cell. Nature 2023, 620 (7972), 122– 127,  DOI: 10.1038/s41586-023-06288-x
      There is no corresponding record for this reference.
    157. 157
      Strotz, L. C.; Simões, M.; Girard, M. G.; Breitkreuz, L.; Kimmig, J.; Lieberman, B. S. Getting Somewhere with the Red Queen: Chasing a Biologically Modern Definition of the Hypothesis. Biol. Lett. 2018, 14 (5), 20170734,  DOI: 10.1098/rsbl.2017.0734
      There is no corresponding record for this reference.
    158. 158
      Solé, R. Revisiting Leigh Van Valen’s “A New Evolutionary Law” (1973). Biol. Theory 2022, 17 (2), 120– 125,  DOI: 10.1007/s13752-021-00391-w
      There is no corresponding record for this reference.
    159. 159
      Hammerling, M. J.; Krüger, A.; Jewett, M. C. Strategies for in Vitro Engineering of the Translation Machinery. Nucleic Acids Res. 2020, 48 (3), 1068– 1083,  DOI: 10.1093/nar/gkz1011
      159
      Strategies for in vitro engineering of the translation machinery
      Hammerling, Michael J.; Krueger, Antje; Jewett, Michael C.
      Nucleic Acids Research (2020), 48 (3), 1068-1083CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      A review. Engineering the process of mol. translation, or protein biosynthesis, has emerged as a major opportunity in synthetic and chem. biol. to generate novel biol. insights and enable new applications (e.g. designer protein therapeutics). Here, we review methods for engineering the process of translation in vitro. We discuss the advantages and drawbacks of the two major strategies-purified and ext.-based systems-and how they may be used to manipulate and study translation. Techniques to engineer each component of the translation machinery are covered in turn, including tRNAs, translation factors, and the ribosome. Finally, future directions and enabling technol. advances for the field are discussed.
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    160. 160
      Williams, T. C.; Averesch, N. J. H.; Winter, G.; Plan, M. R.; Vickers, C. E.; Nielsen, L. K.; Krömer, J. O. Quorum-Sensing Linked RNA Interference for Dynamic Metabolic Pathway Control in Saccharomyces Cerevisiae. Metab. Eng. 2015, 29, 124– 134,  DOI: 10.1016/j.ymben.2015.03.008
      160
      Quorum-sensing linked RNA interference for dynamic metabolic pathway control in Saccharomyces cerevisiae
      Williams, T. C.; Averesch, N. J. H.; Winter, G.; Plan, M. R.; Vickers, C. E.; Nielsen, L. K.; Kromer, J. O.
      Metabolic Engineering (2015), 29 (), 124-134CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)
      Some of the most productive metabolic engineering strategies involve genetic modifications that cause severe metabolic burden on the host cell. Growth-limiting genetic modifications can be more effective if they are 'switched on' after a population growth phase has been completed. To address this problem we have engineered dynamic regulation using a previously developed synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit autonomously triggers gene expression at a high population d., and was linked with an RNA interference module to enable target gene silencing. As a demonstration the circuit was used to control flux through the shikimate pathway for the prodn. of para-hydroxybenzoic acid (PHBA). Dynamic RNA repression allowed gene knock-downs which were identified by elementary flux mode anal. as highly productive but with low biomass formation to be implemented after a population growth phase, resulting in the highest published PHBA titer in yeast (1.1 mM).
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    161. 161
      Robinson, A. O.; Venero, O. M.; Adamala, K. P. Toward Synthetic Life: Biomimetic Synthetic Cell Communication. Curr. Opin. Chem. Biol. 2021, 64, 165– 173,  DOI: 10.1016/j.cbpa.2021.08.008
      161
      Toward synthetic life: Biomimetic synthetic cell communication
      Robinson, Abbey O.; Venero, Orion M.; Adamala, Katarzyna P.
      Current Opinion in Chemical Biology (2021), 64 (), 165-173CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. Engineering synthetic minimal cells provide a controllable chassis for studying the biochem. principles of natural life, increasing our understanding of complex biol. processes. Recently, synthetic cell engineering has enabled communication between both natural live cells and other synthetic cells. A system such as these enable studying interactions between populations of cells, both natural and artificial, and engineering small mol. cell communication protocols for a variety of basic research and practical applications. In this review, we summarize recent progress in engineering communication between synthetic and natural cells, and we speculate about the possible future directions of this work.
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    162. 162
      Garamella, J.; Majumder, S.; Liu, A. P.; Noireaux, V. An Adaptive Synthetic Cell Based on Mechanosensing, Biosensing, and Inducible Gene Circuits. ACS Synth. Biol. 2019, 8 (8), 1913– 1920,  DOI: 10.1021/acssynbio.9b00204
      162
      An Adaptive Synthetic Cell Based on Mechanosensing, Biosensing, and Inducible Gene Circuits
      Garamella, Jonathan; Majumder, Sagardip; Liu, Allen P.; Noireaux, Vincent
      ACS Synthetic Biology (2019), 8 (8), 1913-1920CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      The bottom-up assembly of synthetic cell systems capable of recapitulating biol. functions has become a means to understand living matter by construction. The integration of biomol. components into active, cell-sized, genetically programmed compartments remains, however, a major bottleneck for building synthetic cells. A primary feature of real cells is their ability to actively interact with their surroundings, particularly in stressed conditions. Here, we construct a synthetic cell equipped with an inducible genetic circuit that responds to changes in osmotic pressure through the mechanosensitive channel MscL. Liposomes loaded with an E. coli cell-free transcription-translation (TXTL) system are induced with IPTG when exposed to hypo-osmotic soln., resulting in the expression of a bacterial cytoskeletal protein MreB. MreB assocs. with the membrane to generate a cortex-like structure. Our work provides the first example of mol. integration that couples mechanosensitivity, gene expression, and self-assembly at the inner membrane of synthetic cells.
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    163. 163
      Liu, H.; Yang, Q.; Peng, R.; Kuai, H.; Lyu, Y.; Pan, X.; Liu, Q.; Tan, W. Artificial Signal Feedback Network Mimicking Cellular Adaptivity. J. Am. Chem. Soc. 2019, 141 (16), 6458– 6461,  DOI: 10.1021/jacs.8b13816
      163
      Artificial Signal Feedback Network Mimicking Cellular Adaptivity
      Liu, Hui; Yang, Qiuxia; Peng, Ruizi; Kuai, Hailan; Lyu, Yifan; Pan, Xiaoshu; Liu, Qiaoling; Tan, Weihong
      Journal of the American Chemical Society (2019), 141 (16), 6458-6461CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Inspired by this elegant system of cellular adaptivity, we herein report the rational design of a dynamic artificial adaptive system able to sense and respond to environmental stresses in a unique sense-and-respond mode. Utilizing DNA nanotechnol., we constructed an artificial signal feedback network and anchored it to surface membrane of a model giant membrane vesicle (GMV) protocell. Such system would need to both sense incoming stimuli and emit a feedback response to eliminate the stimuli. To accomplish this mechanistically, our DNA-based artificial signal system, hereinafter termed DASsys, was equipped with a DNA trig-ger-induced DNA polymer formation and dissocn. machinery. Thus, through a sequential cascade of stimulus-induced DNA strand displacement, DASsys could effectively sense and respond to incoming stimuli. Then, by eliminating the stimulus, the membrane surface would return to its initial state, realizing the formation of a cyclical feedback mechanism. Overall, our strategy opens up a route to the construction of artificial signaling system capable of maintaining homeostasis in the cellular micromilieu, and addresses important emerging challenges in bioinspired engineering.
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    164. 164
      Gispert, I.; Hindley, J. W.; Pilkington, C. P.; Shree, H.; Barter, L. M. C.; Ces, O.; Elani, Y. Stimuli-Responsive Vesicles as Distributed Artificial Organelles for Bacterial Activation. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 (42), e2206563119,  DOI: 10.1073/pnas.2206563119
      There is no corresponding record for this reference.
    165. 165
      Aufinger, L.; Simmel, F. C. Establishing Communication Between Artificial Cells. Chemistry 2019, 25 (55), 12659– 12670,  DOI: 10.1002/chem.201901726
      165
      Establishing Communication Between Artificial Cells
      Aufinger, Lukas; Simmel, Friedrich C.
      Chemistry - A European Journal (2019), 25 (55), 12659-12670CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Communication between artificial cells is essential for the realization of complex dynamical behaviors at the multi-cell level. It is also an important prerequisite for modular systems design, because it dets. how spatially sepd. functional modules can coordinate their actions. Among others, mol. communication is required for artificial cell signaling, synchronization of cellular behaviors, computation, group-level decision-making processes and pattern formation in artificial tissues. In this review, an overview of various recent approaches to create communicating artificial cellular systems is provided. In this context, important physicochem. boundary conditions that have to be considered for the design of the communicating cells are also described, and a survey of the most striking emergent behaviors that may be achieved in such systems is given.
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    166. 166
      Pereyre, S.; Sirand-Pugnet, P.; Beven, L.; Charron, A.; Renaudin, H.; Barré, A.; Avenaud, P.; Jacob, D.; Couloux, A.; Barbe, V.; de Daruvar, A.; Blanchard, A.; Bébéar, C. Life on Arginine for Mycoplasma Hominis: Clues from Its Minimal Genome and Comparison with Other Human Urogenital Mycoplasmas. PLoS Genet. 2009, 5 (10), e1000677,  DOI: 10.1371/journal.pgen.1000677
      There is no corresponding record for this reference.
    167. 167
      Woese, C. R.; Maniloff, J.; Zablen, L. B. Phylogenetic Analysis of the Mycoplasmas. Proc. Natl. Acad. Sci. U. S. A. 1980, 77 (1), 494– 498,  DOI: 10.1073/pnas.77.1.494
      There is no corresponding record for this reference.
    168. 168
      Li, S.; Guan, J.-L.; Chien, S. Biochemistry and Biomechanics of Cell Motility. Annu. Rev. Biomed. Eng. 2005, 7, 105– 150,  DOI: 10.1146/annurev.bioeng.7.060804.100340
      168
      Biochemistry and biomechanics of cell motility
      Li, Song; Guan, Jun-Lin; Chien, Shu
      Annual Review of Biomedical Engineering (2005), 7 (), 105-150, 2 platesCODEN: ARBEF7; ISSN:1523-9829. (Annual Reviews Inc.)
      A review. Cell motility is an essential cellular process for a variety of biol. events. The process of cell migration requires the integration and coordination of complex biochem. and biomech. signals. The protrusion force at the leading edge of a cell is generated by the cytoskeleton, and this force generation is controlled by multiple signaling cascades. The formation of new adhesions at the front and the release of adhesions at the rear involve the outside-in and inside-out signaling mediated by integrins and other adhesion receptors. The traction force generated by the cell on the extracellular matrix (ECM) regulates cell-ECM adhesions, and the counter force exerted by ECM on the cell drives the migration. The polarity of cell migration can be amplified and maintained by the feedback loop between the cytoskeleton and cell-ECM adhesions. Cell migration in 3-dimensional ECM has characteristics distinct from that on 2-dimensional ECM. The migration of cells is initiated and modulated by external chem. and mech. factors, such as chemoattractants and the mech. forces acting on the cells and ECM, as well as the surface d., distribution, topog., and rigidity of the ECM.
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    169. 169
      Brunet, T.; Albert, M.; Roman, W.; Coyle, M. C.; Spitzer, D. C.; King, N. A Flagellate-to-Amoeboid Switch in the Closest Living Relatives of Animals. eLife 2021,  DOI: 10.7554/eLife.61037
      There is no corresponding record for this reference.
    170. 170
      Siton-Mendelson, O.; Bernheim-Groswasser, A. Toward the Reconstitution of Synthetic Cell Motility. Cell Adh. Migr. 2016, 10 (5), 461– 474,  DOI: 10.1080/19336918.2016.1170260
      170
      Toward the reconstitution of synthetic cell motility
      Siton-Mendelson Orit; Bernheim-Groswasser Anne
      Cell adhesion & migration (2016), 10 (5), 461-474 ISSN:.
      Cellular motility is a fundamental process essential for embryonic development, wound healing, immune responses, and tissues development. Cells are mostly moving by crawling on external, or inside, substrates which can differ in their surface composition, geometry, and dimensionality. Cells can adopt different migration phenotypes, e.g., bleb-based and protrusion-based, depending on myosin contractility, surface adhesion, and cell confinement. In the few past decades, research on cell motility has focused on uncovering the major molecular players and their order of events. Despite major progresses, our ability to infer on the collective behavior from the molecular properties remains a major challenge, especially because cell migration integrates numerous chemical and mechanical processes that are coupled via feedbacks that span over large range of time and length scales. For this reason, reconstituted model systems were developed. These systems allow for full control of the molecular constituents and various system parameters, thereby providing insight into their individual roles and functions. In this review we describe the various reconstituted model systems that were developed in the past decades. Because of the multiple steps involved in cell motility and the complexity of the overall process, most of the model systems focus on very specific aspects of the individual steps of cell motility. Here we describe the main advancement in cell motility reconstitution and discuss the main challenges toward the realization of a synthetic motile cell.
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    171. 171
      Blanken, D.; van Nies, P.; Danelon, C. Quantitative Imaging of Gene-Expressing Liposomes Reveals Rare Favorable Phenotypes. Phys. Biol. 2019, 16 (4), 045002,  DOI: 10.1088/1478-3975/ab0c62
      171
      Quantitative imaging of gene-expressing liposomes reveals rare favorable phenotypes
      Blanken, Duco; van Nies, Pauline; Danelon, Christophe
      Physical Biology (2019), 16 (4), 045002CODEN: PBHIAT; ISSN:1478-3975. (IOP Publishing Ltd.)
      We report on direct imaging of tens of thousands of gene-expressing liposomes per sample allowing us to assess sub-population features in a statistically relevant manner. Both the vesicle size(diam. <10μm) and lipid compn. (mixt. of phospholipids with zwitterionic and neg. charged headgroups, including cardiolipin) are compatible with the properties of bacterial cells. Therefore, our liposomes provide suitable chassis to host Escherichia coli-derived PURE translation machinery and other bacterial processes in future developments. The potential of high-content imaging to identify rare phenotypes is demonstrated by fact that a subset of the liposome population exhibits remarkably high yield of synthesized protein or prolonged expression lifespan that surpasses performance of ensemble liposome-averaged and bulk reactions. Among the three com. PURE systems tested, PUREfrex2.0 offers the most favorable phenotypes displaying both high yield and long protein synthesis lifespan. Moreover, probing membrane permeability reveals a large heterogeneity amongst liposomes. In situ expression and membrane embedding of the pore-forming connexin leads to a characteristic permeability time profile, while increasing the fraction of permeable liposomes in the population. We see diversity in gene expression dynamics and membrane permeability as an opportunity to complement a rational design approach aiming at further implementing biol. functions in liposome-based synthetic cells.
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    172. 172
      Stano, P.; D’Aguanno, E.; Bolz, J.; Fahr, A.; Luisi, P. L. A Remarkable Self-Organization Process as the Origin of Primitive Functional Cells. Angew. Chem., Int. Ed. Engl. 2013, 52 (50), 13397– 13400,  DOI: 10.1002/anie.201306613
      There is no corresponding record for this reference.
    173. 173
      Weitz, M.; Kim, J.; Kapsner, K.; Winfree, E.; Franco, E.; Simmel, F. C. Diversity in the Dynamical Behaviour of a Compartmentalized Programmable Biochemical Oscillator. Nat. Chem. 2014, 6 (4), 295– 302,  DOI: 10.1038/nchem.1869
      173
      Diversity in the dynamical behaviour of a compartmentalized programmable biochemical oscillator
      Weitz, Maximilian; Kim, Jongmin; Kapsner, Korbinian; Winfree, Erik; Franco, Elisa; Simmel, Friedrich C.
      Nature Chemistry (2014), 6 (4), 295-302CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      In vitro compartmentalization of biochem. reaction networks is a crucial step towards engineering artificial cell-scale devices and systems. At this scale the dynamics of mol. systems becomes stochastic, which introduces several engineering challenges and opportunities. Here we study a programmable transcriptional oscillator system that is compartmentalized into microemulsion droplets with vols. between 33 fl and 16 pl. Simultaneous measurement of large populations of droplets reveals major variations in the amplitude, frequency and damping of the oscillations. Variability increases for smaller droplets and depends on the operating point of the oscillator. Rather than reflecting the stochastic kinetics of the chem. reaction network itself, the variability can be attributed to the statistical variation of reactant concns. created during their partitioning into droplets. We anticipate that robustness to partitioning variability will be a crit. challenge for engineering cell-scale systems, and that highly parallel time-series acquisition from microemulsion droplets will become a key tool for characterization of stochastic circuit function.
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    174. 174
      Hansen, M. M. K.; Meijer, L. H. H.; Spruijt, E.; Maas, R. J. M.; Rosquelles, M. V.; Groen, J.; Heus, H. A.; Huck, W. T. S. Macromolecular Crowding Creates Heterogeneous Environments of Gene Expression in Picolitre Droplets. Nat. Nanotechnol. 2016, 11 (2), 191– 197,  DOI: 10.1038/nnano.2015.243
      174
      Macromolecular crowding creates heterogeneous environments of gene expression in picolitre droplets
      Hansen, Maike M. K.; Meijer, Lenny H. H.; Spruijt, Evan; Maas, Roel J. M.; Rosquelles, Marta Ventosa; Groen, Joost; Heus, Hans A.; Huck, Wilhelm T. S.
      Nature Nanotechnology (2016), 11 (2), 191-197CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      Understanding the dynamics of complex enzymic reactions in highly crowded small vols. is crucial for the development of synthetic minimal cells. Compartmentalized biochem. reactions in cell-sized containers exhibit a degree of randomness due to the small no. of mols. involved. However, it is unknown how the phys. environment contributes to the stochastic nature of multistep enzymic processes. Here, we present a robust method to quantify gene expression noise in vitro using droplet microfluidics. We study the changes in stochasticity in the cell-free gene expression of two genes compartmentalized within droplets as a function of DNA copy no. and macromol. crowding. We find that decreased diffusion caused by a crowded environment leads to the spontaneous formation of heterogeneous microenvironments of mRNA as local prodn. rates exceed the diffusion rates of macromols. This heterogeneity leads to a higher probability of the mol. machinery staying in the same microenvironment, directly increasing the system's stochasticity.
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    175. 175
      Rao, C. V. Expanding the Synthetic Biology Toolbox: Engineering Orthogonal Regulators of Gene Expression. Curr. Opin. Biotechnol. 2012, 23 (5), 689– 694,  DOI: 10.1016/j.copbio.2011.12.015
      There is no corresponding record for this reference.
    176. 176
      Yelleswarapu, M.; van der Linden, A. J.; van Sluijs, B.; Pieters, P. A.; Dubuc, E.; de Greef, T. F. A.; Huck, W. T. S. Sigma Factor-Mediated Tuning of Bacterial Cell-Free Synthetic Genetic Oscillators. ACS Synth. Biol. 2018, 7 (12), 2879– 2887,  DOI: 10.1021/acssynbio.8b00300
      176
      Sigma factor-mediated tuning of bacterial cell-free synthetic genetic oscillators
      Yelleswarapu, Maaruthy; van der Linden, Ardjan J.; van Sluijs, Bob; Pieters, Pascal A.; Dubuc, Emilien; de Greef, Tom F. A.; Huck, Wilhelm T. S.
      ACS Synthetic Biology (2018), 7 (12), 2879-2887CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Cell-free transcription-translation provides a simplified prototyping environment to rapidly design and study synthetic networks. Despite the presence of a well-characterized toolbox of genetic elements, examples of genetic networks that exhibit complex temporal behavior are scarce. Here, we present a genetic oscillator implemented in an Escherichia coli-based cell-free system under steady-state conditions using microfluidic flow reactors. The oscillator had an activator-repressor motif that utilizes the native transcriptional machinery of E. coli: the RNA polymerase (RNAP) and its assocd. σ-factors. We optimized a kinetic model with exptl. data using an evolutionary algorithm to quantify the key regulatory model parameters. The functional modulation of RNAP was investigated by coupling 2 oscillators driven by competing σ-factors, allowing the modification of network properties by means of passive transcriptional regulation.
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    177. 177
      Li, J.; Zhang, C.; Huang, P.; Kuru, E.; Forster-Benson, E. T. C.; Li, T.; Church, G. M. Dissecting Limiting Factors of the Protein Synthesis Using Recombinant Elements (PURE) System. Translation (Austin) 2017, 5 (1), e1327006,  DOI: 10.1080/21690731.2017.1327006
      There is no corresponding record for this reference.
    178. 178
      Lavickova, B.; Maerkl, S. J. A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System. ACS Synth. Biol. 2019, 8 (2), 455– 462,  DOI: 10.1021/acssynbio.8b00427
      178
      A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System
      Lavickova, Barbora; Maerkl, Sebastian J.
      ACS Synthetic Biology (2019), 8 (2), 455-462CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription-translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.
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    179. 179
      Li, J.; Gu, L.; Aach, J.; Church, G. M. Improved Cell-Free RNA and Protein Synthesis System. PLoS One 2014, 9 (9), e106232,  DOI: 10.1371/journal.pone.0106232
      179
      Improved cell-free RNA and protein synthesis system
      Li, Jun; Gu, Liangcai; Aach, John; Church, George M.
      PLoS One (2014), 9 (9), e106232/1-e106232/11, 11 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Cell-free RNA and protein synthesis (CFPS) is becoming increasingly used for protein prodn. as yields increase and costs decrease. Advances in reconstituted CFPS systems such as the Protein synthesis Using Recombinant Elements (PURE) system offer new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, protein microarrays, isotopic labeling, and incorporating unnatural amino acids. In this study, using firefly luciferase synthesis as a reporter system, we improved PURE system productivity up to 5 fold by adding or adjusting a variety of factors that affect transcription and translation, including Elongation factors (EF-Ts, EF-Tu, EF-G and EF4), ribosome recycling factor (RRF), release factors (RF1, RF2, RF3), chaperones (GroEL/ES), BSA and tRNAs. The work provides a more efficient defined in vitro transcription and translation system and a deeper understanding of the factors that limit the whole system efficiency.
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    180. 180
      Jewett, M. C.; Calhoun, K. A.; Voloshin, A.; Wuu, J. J.; Swartz, J. R. An Integrated Cell-Free Metabolic Platform for Protein Production and Synthetic Biology. Mol. Syst. Biol. 2008, 4, 220,  DOI: 10.1038/msb.2008.57
      180
      An integrated cell-free metabolic platform for protein production and synthetic biology
      Jewett Michael C; Calhoun Kara A; Voloshin Alexei; Wuu Jessica J; Swartz James R
      Molecular systems biology (2008), 4 (), 220 ISSN:.
      Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements.
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    181. 181
      Silverman, A. D.; Karim, A. S.; Jewett, M. C. Cell-Free Gene Expression: An Expanded Repertoire of Applications. Nat. Rev. Genet. 2020, 21 (3), 151– 170,  DOI: 10.1038/s41576-019-0186-3
      181
      Cell-free gene expression: an expanded repertoire of applications
      Silverman, Adam D.; Karim, Ashty S.; Jewett, Michael C.
      Nature Reviews Genetics (2020), 21 (3), 151-170CODEN: NRGAAM; ISSN:1471-0056. (Nature Research)
      Cell-free biol. is the activation of biol. processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent tech. renaissance has facilitated high-yielding (grams of protein per L), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biol. design. These advances provide exciting opportunities to profoundly transform synthetic biol. by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compds., on-demand biomanufg., building cells from the bottom up, and next-generation educational kits.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1OitbbO&md5=e227ddcf47b0b961b2b423d414fa3795
    182. 182
      Ouyang, X.; Zhou, X.; Lai, S. N.; Liu, Q.; Zheng, B. Immobilization of Proteins of Cell Extract to Hydrogel Networks Enhances the Longevity of Cell-Free Protein Synthesis and Supports Gene Networks. ACS Synth. Biol. 2021, 10 (4), 749– 755,  DOI: 10.1021/acssynbio.0c00541
      There is no corresponding record for this reference.
    183. 183
      New England Biolabs. NEBExpress Cell-free ecoli Protein Synthesis System. https://www.neb.com/products/e5360-nebexpress-cell-free-ecoli-protein-synthesis-system (accessed on August 21, 2023).
      There is no corresponding record for this reference.
    184. 184
      Zimmerman, S. B.; Trach, S. O. Estimation of Macromolecule Concentrations and Excluded Volume Effects for the Cytoplasm of Escherichia Coli. J. Mol. Biol. 1991, 222 (3), 599– 620,  DOI: 10.1016/0022-2836(91)90499-V
      184
      Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli
      Zimmerman, Steven B.; Trach, Stefan O.
      Journal of Molecular Biology (1991), 222 (3), 599-620CODEN: JMOBAK; ISSN:0022-2836.
      The very high concn. of macromols. within cells can potentially have an overwhelming effect on the thermodn. activity of cellular components because of excluded vol. effects. To est. the magnitudes of such effects, an exptl. study was made of the cytoplasm of Escherichia coli. Parameters from cells and cell exts. are used to calc. approx. activity coeffs. for cytoplasmic conditions. These calcns. require a representation of the sizes, concns. and effective sp. vol. of the macromols. in the exts. Macromol. size representations are obtained either by applying a two-phase distribution assay to define a related homogeneous soln. or by using the mol. mass distribution of macromols. from gel filtration. Macromol. concns. in cytoplasm are obtained from analyses of exts. by applying a correction for the diln. that occurs during extn. That factor is detd. from expts. based upon the known impermeability of the cytoplasmic vol. to sucrose in intact E. coli. Macromol. concns. in the cytoplasm of E. coli in either exponential or stationary growth phase are estd. to be ≈0.3 to 0.4 g/mL. Macromol. sp. vol. are inferred from the compn. of close-packed ppts. induced by polyethylene glycol. Several well-characterized proteins which bind to DNA (lac repressor, RNA polymerase) are extremely sensitive to changes in salt concn. in studies in vitro, but are insensitive in studies in vivo. Application of the activity coeffs. from the present work indicates that at least part of this discrepancy arises from the differences in excluded vols. in these studies. Applications of the activity coeffs. to soly. or to assocn. reactions are also discussed, as are changes assocd. with cell growth phase and osmotic or other effects. The use of solns. of purified macromols. that emulate the crowding conditions inferred for cytoplasm is discussed.
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    185. 185
      Record, M. T., Jr; Courtenay, E. S.; Cayley, D. S.; Guttman, H. J. Responses of E. Coli to Osmotic Stress: Large Changes in Amounts of Cytoplasmic Solutes and Water. Trends Biochem. Sci. 1998, 23 (4), 143– 148,  DOI: 10.1016/S0968-0004(98)01196-7
      185
      Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water
      Record, M. Thomas, Jr.; Courtenay, Elizabeth S.; Cayley, D. Scott; Guttman, Harry J.
      Trends in Biochemical Sciences (1998), 23 (4), 143-148CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Science Ltd.)
      A review with 37 refs. Escherichia coli is capable of growing in environments ranging from very dil. aq. solns. of essential nutrients to media contg. molar concns. of salts or nonelectrolyte solutes. Growth in environments with such a wide range (at least 100-fold) of osmolarities poses significant physiol. challenges for cells. To meet these challenges, E. coli adjusts a wide range of cytoplasmic soln. variables, including the cytoplasmic amts. both of water and of charged and uncharged solutes.
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    186. 186
      Cayley, S.; Lewis, B. A.; Guttman, H. J.; Record, M. T. Jr. Characterization of the Cytoplasm of Escherichia Coli K-12 as a Function of External Osmolarity. Implications for Protein-DNA Interactions in Vivo. J. Mol. Biol. 1991, 222 (2), 281– 300,  DOI: 10.1016/0022-2836(91)90212-O
      There is no corresponding record for this reference.
    187. 187
      Li, J.; Haas, W.; Jackson, K.; Kuru, E.; Jewett, M. C.; Fan, Z. H.; Gygi, S.; Church, G. M. Cogenerating Synthetic Parts toward a Self-Replicating System. ACS Synth. Biol. 2017, 6 (7), 1327– 1336,  DOI: 10.1021/acssynbio.6b00342
      187
      Co-generating Synthetic Parts toward a Self-Replicating System
      Li, Jun; Haas, Wilhelm; Jackson, Kirsten; Kuru, Erkin; Jewett, Michael C.; Fan, Z. Hugh; Gygi, Steven; Church, George M.
      ACS Synthetic Biology (2017), 6 (7), 1327-1336CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      To build replicating systems with new functions, the engineering of existing biol. machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE might be the basis for a radically alterable, lifelike system after optimization. Here, the authors regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. The authors show that using stable isotope labeling with amino acids, mass spectrometry based quant. proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when coexpressed in batch format PURE system. By optimizing DNA template concns. and adapting a miniaturized Fluid Array Device with optimized feeding soln., the authors were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in coexpression pool varied from ∼1.5 to 5-fold compared to the batch mode, with up to ∼2.4 μM yield for a single r-protein. Reconstituted ribosomes under physiol. condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ∼13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. This work also points to what is still needed to obtain self-replicating synthetic ribosomes in situ in the PURE system.
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    188. 188
      Murase, Y.; Nakanishi, H.; Tsuji, G.; Sunami, T.; Ichihashi, N. In Vitro Evolution of Unmodified 16S rRNA for Simple Ribosome Reconstitution. ACS Synth. Biol. 2018, 7 (2), 576– 583,  DOI: 10.1021/acssynbio.7b00333
      There is no corresponding record for this reference.
    189. 189
      Jewett, M. C.; Fritz, B. R.; Timmerman, L. E.; Church, G. M. In Vitro Integration of Ribosomal RNA Synthesis, Ribosome Assembly, and Translation. Mol. Syst. Biol. 2013, 9, 678,  DOI: 10.1038/msb.2013.31
      189
      In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation
      Jewett, Michael C.; Fritz, Brian R.; Timmerman, Laura E.; Church, George M.
      Molecular Systems Biology (2013), 9 (), 678CODEN: MSBOC3; ISSN:1744-4292. (Nature Publishing Group)
      Purely in vitro ribosome synthesis could provide a crit. step towards unraveling the systems biol. of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli exts. While conventional methods for E. coli ribosome reconstitution are non-physiol., our approach attempts to mimic chem. conditions in the cytoplasm, thus permitting several biol. processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technol. enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties.
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    190. 190
      Fritz, B. R.; Jamil, O. K.; Jewett, M. C. Implications of Macromolecular Crowding and Reducing Conditions for in Vitro Ribosome Construction. Nucleic Acids Res. 2015, 43 (9), 4774– 4784,  DOI: 10.1093/nar/gkv329
      190
      Implications of macromolecular crowding and reducing conditions for in vitro ribosome construction
      Fritz, Brian R.; Jamil, Osman K.; Jewett, Michael C.
      Nucleic Acids Research (2015), 43 (9), 4774-4784CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      The in vitro construction of Escherichia coli ribosomes could elucidate a deeper understanding of these complex mol. machines and make possible the prodn. of synthetic variants with new functions. Toward this goal, the authors recently developed an integrated synthesis, assembly, and translation (iSAT) system that allows for co-activation of rRNA transcription and ribosome assembly, mRNA transcription, and protein translation without intact cells. Here, the authors discovered that macromol. crowding and reducing agents increased overall iSAT protein synthesis; the combination of 6% Ficoll 400 and 2 mM DTBA yielded an approx. 5-fold increase in overall iSAT protein synthesis activity. By utilizing a fluorescent RNA aptamer, fluorescent reporter proteins, and ribosome sedimentation anal., the authors showed that crowding agents increased iSAT yields by enhancing translation while reducing agents increased rRNA transcription and ribosome assembly. Finally, the authors showed that iSAT ribosomes possessed ∼70% of the protein synthesis activity of in vivo-assembled E. coli ribosomes. This work improved iSAT protein synthesis through the addn. of crowding and reducing agents, provides a thorough understanding of the effect of these additives within the iSAT system and demonstrated how iSAT allows for manipulation and anal. of ribosome biogenesis in the context of an in vitro transcription-translation system.
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    191. 191
      Hammerling, M. J.; Fritz, B. R.; Yoesep, D. J.; Kim, D. S.; Carlson, E. D.; Jewett, M. C. In Vitro Ribosome Synthesis and Evolution through Ribosome Display. Nat. Commun. 2020, 11 (1), 1108,  DOI: 10.1038/s41467-020-14705-2
      191
      In-vitro ribosome synthesis and evolution through ribosome display
      Hammerling, Michael J.; Fritz, Brian R.; Yoesep, Danielle J.; Kim, Do Soon; Carlson, Erik D.; Jewett, Michael C.
      Nature Communications (2020), 11 (1), 1108CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in-vitro methodol. for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ∼1.7 X 107 member library of rRNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ∼4 X 103 rRNA variants. We further demonstrate the prevalence of pos. epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of mol. translation and enable selection of ribosomes with altered properties.
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    192. 192
      Wang, T.; Lu, Y. Toward Minimal Transcription-Translation Machinery. ACS Synth. Biol. 2023, 12 (11), 3312– 3327,  DOI: 10.1021/acssynbio.3c00324
      There is no corresponding record for this reference.
    193. 193
      Oberholzer, T.; Albrizio, M.; Luisi, P. L. Polymerase Chain Reaction in Liposomes. Chem. Biol. 1995, 2 (10), 677– 682,  DOI: 10.1016/1074-5521(95)90031-4
      193
      Polymerase chain reaction in liposomes
      Oberholzer, Thomas; Albrizio, Maria; Luisi, Pier Luigi
      Chemistry & Biology (1995), 2 (10), 677-82CODEN: CBOLE2; ISSN:1074-5521. (Current Biology)
      Background: Compartmentalization of biochem. reactions within a spherically closed bilayer is an important step in the mol. evolution of cells. Liposomes are the most suitable structures to model this kind of chem. We have used the polymerase chain reaction (PCR) to demonstrate that complex biochem. reactions such as DNA replication can be carried out inside these compartments. Results: We describe the first example of DNA amplification by the PCR occurring inside liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or of a mixt. of POPC and phosphatidylserine. We show that these liposomes are stable even under the high temp. conditions used for PCR. Although only a very small fraction of liposomes contains all eight different reagents together, a significant amt. of DNA is produced which can be obsd. by polyacrylamide gel electrophoresis. Conclusions: This work shows that it is possible to carry out complex biochem. reactions within liposomes, which may be germane to the question of the origin of living cells. We have established the parameters and conditions that are crit. for carrying out this complex reaction within the liposome compartment.
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    194. 194
      Kawska, A.; Carvalho, K.; Manzi, J.; Boujemaa-Paterski, R.; Blanchoin, L.; Martiel, J.-L.; Sykes, C. How Actin Network Dynamics Control the Onset of Actin-Based Motility. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (36), 14440– 14445,  DOI: 10.1073/pnas.1117096109
      There is no corresponding record for this reference.
    195. 195
      Hürtgen, D.; Härtel, T.; Murray, S. M.; Sourjik, V.; Schwille, P. Functional Modules of Minimal Cell Division for Synthetic Biology. Adv. Biosyst. 2019, 3 (6), e1800315,  DOI: 10.1002/adbi.201800315
      There is no corresponding record for this reference.
    196. 196
      Pelletier, J. F.; Sun, L.; Wise, K. S.; Assad-Garcia, N.; Karas, B. J.; Deerinck, T. J.; Ellisman, M. H.; Mershin, A.; Gershenfeld, N.; Chuang, R.-Y.; Glass, J. I.; Strychalski, E. A. Genetic Requirements for Cell Division in a Genomically Minimal Cell. Cell 2021, 184 (9), 2430– 2440,  DOI: 10.1016/j.cell.2021.03.008
      196
      Genetic requirements for cell division in a genomically minimal cell
      Pelletier, James F.; Sun, Lijie; Wise, Kim S.; Assad-Garcia, Nacyra; Karas, Bogumil J.; Deerinck, Thomas J.; Ellisman, Mark H.; Mershin, Andreas; Gershenfeld, Neil; Chuang, Ray-Yuan; Glass, John I.; Strychalski, Elizabeth A.
      Cell (Cambridge, MA, United States) (2021), 184 (9), 2430-2440.e16CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiol. processes. Although this minimal cell includes genes essential for population growth, the physiol. of its single cells remained uncharacterized. To investigate striking morphol. variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and det. genes affecting cell morphol. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphol. similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-assocd. proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphol. in a genomically minimal cell. The fully annotated genome sequence of JCVI-syn3A is deposited in NCBI (GenBank: CP016816.2).
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    197. 197
      Zhang, S.; Contini, C.; Hindley, J. W.; Bolognesi, G.; Elani, Y.; Ces, O. Engineering Motile Aqueous Phase-Separated Droplets via Liposome Stabilisation. Nat. Commun. 2021, 12 (1), 1673,  DOI: 10.1038/s41467-021-21832-x
      197
      Engineering motile aqueous phase-separated droplets via liposome stabilisation
      Zhang, Shaobin; Contini, Claudia; Hindley, James W.; Bolognesi, Guido; Elani, Yuval; Ces, Oscar
      Nature Communications (2021), 12 (1), 1673CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      There are increasing efforts to engineer functional compartments that mimic cellular behaviors from the bottom-up. One behavior that is receiving particular attention is motility, due to its biotechnol. potential and ubiquity in living systems. Many existing platforms make use of the Marangoni effect to achieve motion in water/oil (w/o) droplet systems. However, most of these systems are unsuitable for biol. applications due to biocompatibility issues caused by the presence of oil phases. Here we report a biocompatible all aq. (wt./wt.) PEG/dextran Pickering-like emulsion system consisting of liposome-stabilized cell-sized droplets, where the stability can be easily tuned by adjusting liposome compn. and concn. We demonstrate that the compartments are capable of neg. chemotaxis: these droplets can respond to a PEG/dextran polymer gradient through directional motion down to the gradient. The biocompatibility, motility and partitioning abilities of this droplet system offers new directions to pursue research in motion-related biol. processes.
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    198. 198
      Li, M.; Brinkmann, M.; Pagonabarraga, I.; Seemann, R.; Fleury, J.-B. Spatiotemporal Control of Cargo Delivery Performed by Programmable Self-Propelled Janus Droplets. Commun. Phys. 2018, 1 (1), 1– 8,  DOI: 10.1038/s42005-018-0025-4
      198
      Breakdown of diffusivity-entropy scaling in colloidal glass-forming liquids
      Li, Bo; Xiao, Xiuming; Lou, Kai; Wang, Shuxia; Wen, Weijia; Wang, Ziren
      Communications Physics (2018), 1 (1), 1-9CODEN: CPOHDJ; ISSN:2399-3650. (Nature Research)
      Glass is a liq. that has lost its ability to flow. Why this particular substance undergoes such a dramatic kinetic slowdown yet remains barely distinguishable in structure from its fluid state upon cooling constitutes the central question of glass transition physics. Here, we investigate the pathway of kinetic slowdown in glass-forming liqs. that consist of monolayers of ellipsoidal or binary spherical colloids. In contrast to rotational motion, the dynamics of the translational motion begin to violently slow down at considerably low area fractions (.vphi.T). At .vphi.T, anomalous translation-rotation coupling is enhanced and the topog. of the free energy landscape become rugged. Based on the pos. correlation between .vphi.T and fragility, the measurement of .vphi.T offers a novel method for predicting glassy dynamics, circumventing the prohibitive increase in equil. times required in high-d. regions. Our results highlight the role that thermodynamical entropy plays in glass transitions.
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    199. 199
      Litschel, T.; Kelley, C. F.; Holz, D.; Adeli Koudehi, M.; Vogel, S. K.; Burbaum, L.; Mizuno, N.; Vavylonis, D.; Schwille, P. Reconstitution of Contractile Actomyosin Rings in Vesicles. Nat. Commun. 2021, 12 (1), 2254,  DOI: 10.1038/s41467-021-22422-7
      199
      Reconstitution of contractile actomyosin rings in vesicles
      Litschel, Thomas; Kelley, Charlotte F.; Holz, Danielle; Adeli Koudehi, Maral; Vogel, Sven K.; Burbaum, Laura; Mizuno, Naoko; Vavylonis, Dimitrios; Schwille, Petra
      Nature Communications (2021), 12 (1), 2254CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      One of the grand challenges of bottom-up synthetic biol. is the development of minimal machineries for cell division. The mech. transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the mol. scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theor. modeling. By changing few key parameters, actin polymn. can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theor. considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.
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    200. 200
      Bashirzadeh, Y.; Moghimianavval, H.; Liu, A. P. Encapsulated Actomyosin Patterns Drive Cell-like Membrane Shape Changes. iScience 2022, 25 (5), 104236,  DOI: 10.1016/j.isci.2022.104236
      200
      Encapsulated actomyosin patterns drive cell-like membrane shape changes
      Bashirzadeh, Yashar; Moghimianavval, Hossein; Liu, Allen P.
      iScience (2022), 25 (5), 104236CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)
      Cell shape changes from locomotion to cytokinesis are, to a large extent, driven by myosin-driven remodeling of cortical actin patterns. Passive crosslinkers such as α-actinin and fascin as well as actin nucleator Arp2/3 complex largely det. actin network architecture and, consequently, membrane shape changes. Here we reconstitute actomyosin networks inside cell-sized lipid bilayer vesicles and show that depending on vesicle size and concns. of α-actinin and fascin actomyosin networks assemble into ring and aster-like patterns. Anchoring actin to the membrane does not change actin network architecture yet exerts forces and deforms the membrane when assembled in the form of a contractile ring. In the presence of α-actinin and fascin, an Arp2/3 complex-mediated actomyosin cortex is shown to assemble a ring-like pattern at the equatorial cortex followed by myosin-driven clustering and consequently blebbing. An active gel theory unifies a model for the obsd. membrane shape changes induced by the contractile cortex.
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    201. 201
      Gardner, P. M.; Winzer, K.; Davis, B. G. Sugar Synthesis in a Protocellular Model Leads to a Cell Signalling Response in Bacteria. Nat. Chem. 2009, 1 (5), 377– 383,  DOI: 10.1038/nchem.296
      201
      Sugar synthesis in a protocellular model leads to a cell signalling response in bacteria
      Gardner, Paul M.; Winzer, Klaus; Davis, Benjamin G.
      Nature Chemistry (2009), 1 (5), 377-383, S377/1-S377/50CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      The design of systems with life-like properties from simple chem. components may offer insights into biol. processes, with the ultimate goal of creating an artificial chem. cell that would be considered to be alive. Most efforts to create artificial cells have concd. on systems based on complex natural mols. such as DNA and RNA. Here we have constructed a lipid-bound protometabolism that synthesizes complex carbohydrates from simple feedstocks, which are capable of engaging the natural quorum sensing mechanism of the marine bacterium Vibrio harveyi and stimulating a proportional bioluminescent response. This encapsulated system may represent the first step towards the realization of a cellular mimic' and a starting point for bottom-up' designs of other chem. cells, which could perhaps display complex behaviors such as communication with natural cells.
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    202. 202
      Lentini, R.; Santero, S. P.; Chizzolini, F.; Cecchi, D.; Fontana, J.; Marchioretto, M.; Del Bianco, C.; Terrell, J. L.; Spencer, A. C.; Martini, L.; Forlin, M.; Assfalg, M.; Dalla Serra, M.; Bentley, W. E.; Mansy, S. S. Integrating Artificial with Natural Cells to Translate Chemical Messages That Direct E. Coli Behaviour. Nat. Commun. 2014, 5, 4012,  DOI: 10.1038/ncomms5012
      202
      Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour
      Lentini, Roberta; Santero, Silvia Perez; Chizzolini, Fabio; Cecchi, Dario; Fontana, Jason; Marchioretto, Marta; Del Bianco, Cristina; Terrell, Jessica L.; Spencer, Amy C.; Martini, Laura; Forlin, Michele; Assfalg, Michael; Dalla Serra, Mauro; Bentley, William E.; Mansy, Sheref S.
      Nature Communications (2014), 5 (), 4012CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      Previous efforts to control cellular behavior have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behavior of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chem. communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chem. message that E. coli cannot sense on its own to a mol. that activates a natural cellular response. This methodol. could open new opportunities in engineering cellular behavior without exploiting genetically modified organisms.
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    203. 203
      Ding, Y.; Contreras-Llano, L. E.; Morris, E.; Mao, M.; Tan, C. Minimizing Context Dependency of Gene Networks Using Artificial Cells. ACS Appl. Mater. Interfaces 2018, 10 (36), 30137– 30146,  DOI: 10.1021/acsami.8b10029
      There is no corresponding record for this reference.
    204. 204
      Toparlak, Ö. D.; Zasso, J.; Bridi, S.; Serra, M. D.; Macchi, P.; Conti, L.; Baudet, M.-L.; Mansy, S. S. Artificial Cells Drive Neural Differentiation. Sci. Adv. 2020,  DOI: 10.1126/sciadv.abb4920
      There is no corresponding record for this reference.
    205. 205
      Fanalista, F.; Birnie, A.; Maan, R.; Burla, F.; Charles, K.; Pawlik, G.; Deshpande, S.; Koenderink, G. H.; Dogterom, M.; Dekker, C. Shape and Size Control of Artificial Cells for Bottom-Up Biology. ACS Nano 2019, 13 (5), 5439– 5450,  DOI: 10.1021/acsnano.9b00220
      205
      Shape and Size Control of Artificial Cells for Bottom-Up Biology
      Fanalista, Federico; Birnie, Anthony; Maan, Renu; Burla, Federica; Charles, Kevin; Pawlik, Grzegorz; Deshpande, Siddharth; Koenderink, Gijsje H.; Dogterom, Marileen; Dekker, Cees
      ACS Nano (2019), 13 (5), 5439-5450CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Bottom-up biol. is an expanding research field that aims to understand the mechanisms underlying biol. processes via in vitro assembly of their essential components in synthetic cells. As encapsulation and controlled manipulation of these elements is a crucial step in the recreation of such cell-like objects, microfluidics is increasingly used for the prodn. of minimal artificial containers such as single-emulsion droplets, double-emulsion droplets, and liposomes. Despite the importance of cell morphol. on cellular dynamics, current synthetic-cell studies mainly use spherical containers, and methods to actively shape manipulate these have been lacking. In this paper, the authors describe a microfluidic platform to deform the shape of artificial cells into a variety of shapes (rods and disks) with adjustable cell-like dimensions below 5 μm, thereby mimicking realistic cell morphologies. To illustrate the potential of the method, the authors reconstitute three biol. relevant protein systems (FtsZ, microtubules, collagen) inside rod-shaped containers and study the arrangement of the protein networks inside these synthetic containers with physiol. relevant morphologies resembling those found in living cells.
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    206. 206
      Van de Cauter, L.; Fanalista, F.; van Buren, L.; De Franceschi, N.; Godino, E.; Bouw, S.; Danelon, C.; Dekker, C.; Koenderink, G. H.; Ganzinger, K. A. Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles. ACS Synth. Biol. 2021, 10 (7), 1690– 1702,  DOI: 10.1021/acssynbio.1c00068
      206
      Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles
      Van de Cauter, Lori; Fanalista, Federico; van Buren, Lennard; De Franceschi, Nicola; Godino, Elisa; Bouw, Sharon; Danelon, Christophe; Dekker, Cees; Koenderink, Gijsje H.; Ganzinger, Kristina A.
      ACS Synthetic Biology (2021), 10 (7), 1690-1702CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Giant unilamellar vesicles (GUVs) are often used to mimic biol. membranes in reconstitution expts. They are also widely used in research on synthetic cells, as they provide a mech. responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomols. in GUVs, there is no one-size-fits-all soln. and reliable GUV fabrication still remains a major exptl. hurdle in the field. Here, we show that defect-free GUVs contg. complex biochem. systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of biol. systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (protein synthesis using recombinant elements) system. Our optimized cDICE method displays promising potential to become a std. method in biophysics and bottom-up synthetic biol.
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    207. 207
      Ganar, K. A.; Leijten, L.; Deshpande, S. Actinosomes: Condensate-Templated Containers for Engineering Synthetic Cells. ACS Synth. Biol. 2022, 11 (8), 2869– 2879,  DOI: 10.1021/acssynbio.2c00290
      207
      Actinosomes: Condensate-Templated Containers for Engineering Synthetic Cells
      Ganar, Ketan A.; Leijten, Liza; Deshpande, Siddharth
      ACS Synthetic Biology (2022), 11 (8), 2869-2879CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Engineering synthetic cells has a broad appeal, from understanding living cells to designing novel biomaterials for therapeutics, biosensing, and hybrid interfaces. A key prerequisite to creating synthetic cells is a three-dimensional container capable of orchestrating biochem. reactions. In this study, we present an easy and effective technique to make cell-sized porous containers, coined actinosomes, using the interactions between biomol. condensates and the actin cytoskeleton. This approach uses polypeptide/nucleoside triphosphate condensates and localizes actin monomers on their surface. By triggering actin polymn. and using osmotic gradients, the condensates are transformed into containers, with the boundary made up of actin filaments and polylysine polymers. We show that the guanosine triphosphate (GTP)-to-ATP (ATP) ratio is a crucial parameter for forming actinosomes: insufficient ATP prevents condensate dissoln., while excess ATP leads to undesired crumpling. Permeability studies reveal the porous surface of actinosomes, allowing small mols. to pass through while restricting bigger macromols. within the interior. We show the functionality of actinosomes as bioreactors by carrying out in vitro protein translation within them. Actinosomes are a handy addn. to the synthetic cell platform, with appealing properties like ease of prodn., inherent encapsulation capacity, and a potentially active surface to trigger signaling cascades and form multicellular assemblies, conceivably useful for biotechnol. applications.
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    208. 208
      Frischmon, C.; Sorenson, C.; Winikoff, M.; Adamala, K. P. Build-a-Cell: Engineering a Synthetic Cell Community. Life 2021, 11 (11), 1176,  DOI: 10.3390/life11111176
      There is no corresponding record for this reference.
    209. 209
      Schwille, P.; Spatz, J.; Landfester, K.; Bodenschatz, E.; Herminghaus, S.; Sourjik, V.; Erb, T. J.; Bastiaens, P.; Lipowsky, R.; Hyman, A.; Dabrock, P.; Baret, J.-C.; Vidakovic-Koch, T.; Bieling, P.; Dimova, R.; Mutschler, H.; Robinson, T.; Tang, T.-Y. D.; Wegner, S.; Sundmacher, K. MaxSynBio: Avenues Towards Creating Cells from the Bottom Up. Angew. Chem., Int. Ed. Engl. 2018, 57 (41), 13382– 13392,  DOI: 10.1002/anie.201802288
      209
      MaxSynBio: Avenues Towards Creating Cells from the Bottom Up
      Schwille Petra; Mutschler Hannes; Spatz Joachim; Landfester Katharina; Wegner Seraphine; Bodenschatz Eberhard; Herminghaus Stephan; Sourjik Victor; Erb Tobias J; Bastiaens Philippe; Bieling Peter; Lipowsky Reinhard; Dimova Rumiana; Robinson Tom; Hyman Anthony; Tang T-Y Dora; Dabrock Peter; Baret Jean-Christophe; Vidakovic-Koch Tanja; Sundmacher Kai
      Angewandte Chemie (International ed. in English) (2018), 57 (41), 13382-13392 ISSN:.
      A large German research consortium mainly within the Max Planck Society ("MaxSynBio") was formed to investigate living systems from a fundamental perspective. The research program of MaxSynBio relies solely on the bottom-up approach to synthetic biology. MaxSynBio focuses on the detailed analysis and understanding of essential processes of life through modular reconstitution in minimal synthetic systems. The ultimate goal is to construct a basic living unit entirely from non-living components. The fundamental insights gained from the activities in MaxSynBio could eventually be utilized for establishing a new generation of biotechnological processes, which would be based on synthetic cell constructs that replace the natural cells currently used in conventional biotechnology.
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    210. 210
      Habets, M. G. J. L.; Zwart, H. A. E.; van Est, R. Why the Synthetic Cell Needs Democratic Governance. Trends Biotechnol. 2021, 39 (6), 539– 541,  DOI: 10.1016/j.tibtech.2020.11.006
      There is no corresponding record for this reference.
    211. 211
      Rasmussen, S. Protocells: Bridging Nonliving and Living Matter; MIT Press, 2008.
      There is no corresponding record for this reference.
    212. 212
      Schwille, P. Bottom-up Synthetic Biology: Engineering in a Tinkerer’s World. Science 2011, 333 (6047), 1252– 1254,  DOI: 10.1126/science.1211701
      212
      Bottom-Up Synthetic Biology: Engineering in a Tinkerer's World
      Schwille, Petra
      Science (Washington, DC, United States) (2011), 333 (6047), 1252-1254CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A review. How synthetic can "synthetic biol." be A literal interpretation of the name of this new life science discipline invokes expectations of the systematic construction of biol. systems with cells being built module by module-from the bottom up. But can this possibly be achieved, taking into account the enormous complexity and redundancy of living systems, which distinguish them quite remarkably from design features that characterize human inventions There are several recent developments in biol., in tight conjunction with quant. disciplines, that may bring this literal perspective into the realm of the possible. However, such bottom-up engineering requires tools that were originally designed by nature's greatest tinkerer: evolution.
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    213. 213
      Abil, Z.; Danelon, C. Roadmap to Building a Cell: An Evolutionary Approach. Front. Bioeng. Biotechnol. 2020, 8, 927,  DOI: 10.3389/fbioe.2020.00927
      213
      Roadmap to Building a Cell: An Evolutionary Approach
      Abil Zhanar; Danelon Christophe
      Frontiers in bioengineering and biotechnology (2020), 8 (), 927 ISSN:2296-4185.
      Laboratory synthesis of an elementary biological cell from isolated components may aid in understanding of the fundamental principles of life and will provide a platform for a range of bioengineering and medical applications. In essence, building a cell consists in the integration of cellular modules into system's level functionalities satisfying a definition of life. To achieve this goal, we propose in this perspective to undertake a semi-rational, system's level evolutionary approach. The strategy would require iterative cycles of genetic integration of functional modules, diversification of hereditary information, compartmentalized gene expression, selection/screening, and possibly, assistance from open-ended evolution. We explore the underlying challenges to each of these steps and discuss possible solutions toward the bottom-up construction of an artificial living cell.
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    214. 214
      Yarmuth; [d-Ky-3], J. A. Inflation Reduction Act of 2022 ; 2022. http://www.congress.gov/ (accessed on April 30, 2023).
      There is no corresponding record for this reference.
    215. 215
      The White House. Executive order on advancing biotechnology and biomanufacturing innovation for a sustainable, safe, and secure American bioeconomy. The White House. https://www.whitehouse.gov/briefing-room/presidential-actions/2022/09/12/executive-order-on-advancing-biotechnology-and-biomanufacturing-innovation-for-a-sustainable-safe-and-secure-american-bioeconomy/ (accessed on April 30, 2023).
      There is no corresponding record for this reference.
    216. 216
      BioMADE. https://www.biomade.org/ (accessed on April 30, 2023).
      There is no corresponding record for this reference.
    217. 217
      Huang, A.; Nguyen, P. Q.; Stark, J. C.; Takahashi, M. K.; Donghia, N.; Ferrante, T.; Dy, A. J.; Hsu, K. J.; Dubner, R. S.; Pardee, K.; Jewett, M. C.; Collins, J. J. BioBitsTM Explorer: A Modular Synthetic Biology Education Kit. Sci. Adv. 2018, 4 (8), eaat5105,  DOI: 10.1126/sciadv.aat5105
      There is no corresponding record for this reference.
    218. 218
      Stark, J. C.; Huang, A.; Nguyen, P. Q.; Dubner, R. S.; Hsu, K. J.; Ferrante, T. C.; Anderson, M.; Kanapskyte, A.; Mucha, Q.; Packett, J. S.; Patel, P.; Patel, R.; Qaq, D.; Zondor, T.; Burke, J.; Martinez, T.; Miller-Berry, A.; Puppala, A.; Reichert, K.; Schmid, M.; Brand, L.; Hill, L. R.; Chellaswamy, J. F.; Faheem, N.; Fetherling, S.; Gong, E.; Gonzalzles, E. M.; Granito, T.; Koritsaris, J.; Nguyen, B.; Ottman, S.; Palffy, C.; Patel, A.; Skweres, S.; Slaton, A.; Woods, T.; Donghia, N.; Pardee, K.; Collins, J. J.; Jewett, M. C. BioBitsTM Bright: A Fluorescent Synthetic Biology Education Kit. Sci. Adv. 2018, 4 (8), eaat5107,  DOI: 10.1126/sciadv.aat5107
      There is no corresponding record for this reference.
    219. 219
      Stark, J. C.; Huang, A.; Hsu, K. J.; Dubner, R. S.; Forbrook, J.; Marshalla, S.; Rodriguez, F.; Washington, M.; Rybnicky, G. A.; Nguyen, P. Q.; Hasselbacher, B.; Jabri, R.; Kamran, R.; Koralewski, V.; Wightkin, W.; Martinez, T.; Jewett, M. C. BioBits Health: Classroom Activities Exploring Engineering, Biology, and Human Health with Fluorescent Readouts. ACS Synth. Biol. 2019, 8 (5), 1001– 1009,  DOI: 10.1021/acssynbio.8b00381
      219
      BioBits Health: Classroom Activities Exploring Engineering, Biology, and Human Health with Fluorescent Readouts
      Stark, Jessica C.; Huang, Ally; Hsu, Karen J.; Dubner, Rachel S.; Forbrook, Jason; Marshalla, Suzanne; Rodriguez, Faith; Washington, Mechelle; Rybnicky, Grant A.; Nguyen, Peter Q.; Hasselbacher, Brenna; Jabri, Ramah; Kamran, Rijha; Koralewski, Veronica; Wightkin, Will; Martinez, Thomas; Jewett, Michael C.
      ACS Synthetic Biology (2019), 8 (5), 1001-1009CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Recent advances in synthetic biol. have resulted in biol. technologies with the potential to reshape the way we understand and treat human disease. Educating students about the biol. and ethics underpinning these technologies is crit. to empower them to make informed future policy decisions regarding their use and to inspire the next generation of synthetic biologists. However, hands-on, educational activities that convey emerging synthetic biol. topics can be difficult to implement due to the expensive equipment and expertise required to grow living cells. We present BioBits Health, an educational kit contg. lab activities and supporting curricula for teaching antibiotic resistance mechanisms and CRISPR-Cas9 gene editing in high school classrooms. This kit links complex biol. concepts to visual, fluorescent readouts in user-friendly freeze-dried cell-free reactions. BioBits Health represents a set of educational resources that promises to encourage teaching of cutting-edge, health-related synthetic biol. topics in classrooms and other nonlab. settings.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXmt1Olu70%253D&md5=99551c2017559b0b1275e1d168dfb5a4
    220. 220
      Rybnicky, G. A.; Dixon, R. A.; Kuhn, R. M.; Karim, A. S.; Jewett, M. C. Development of a Freeze-Dried CRISPR-Cas12 Sensor for Detecting Wolbachia in the Secondary Science Classroom. ACS Synth. Biol. 2022, 11 (2), 835– 842,  DOI: 10.1021/acssynbio.1c00503
      There is no corresponding record for this reference.
    221. 221
      Jung, K. J.; Rasor, B. J.; Rybnicky, G. A.; Silverman, A. D.; Standeven, J.; Kuhn, R.; Granito, T.; Ekas, H. M.; Wang, B. M.; Karim, A. S.; Lucks, J. B.; Jewett, M. C. At-Home, Cell-Free Synthetic Biology Education Modules for Transcriptional Regulation and Environmental Water Quality Monitoring. bioRxiv , January 9, 2023.  DOI: 10.1101/2023.01.09.523248 .
      There is no corresponding record for this reference.
    222. 222
      Perry, E.; Weber, J.; Pataranutaporn, P.; Volf, V.; Gonzalez, L. M.; Nejad, S.; Angleton, C.; Chen, J.-E.; Gabo, A.; Jammalamadaka, M. S. S.; Kuru, E.; Fortuna, P.; Rico, A.; Sulich, K.; Wawrzyniak, D.; Jacobson, J.; Church, G.; Kong, D. How to Grow (almost) Anything: A Hybrid Distance Learning Model for Global Laboratory-Based Synthetic Biology Education. Nat. Biotechnol. 2022, 40 (12), 1874– 1879,  DOI: 10.1038/s41587-022-01601-x
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      How to grow (almost) anything: a hybrid distance learning model for global laboratory-based synthetic biology education
      Perry, Eyal; Weber, Jessica; Pataranutaporn, Pat; Volf, Verena; Gonzalez, Laura Maria; Nejad, Sara; Angleton, Carolyn; Chen, Jia-En; Gabo, Ananda; Jammalamadaka, Mani Sai Suryateja; Kuru, Erkin; Fortuna, Patrick; Rico, Andres; Sulich, Karolina; Wawrzyniak, Dominika; Jacobson, Joseph; Church, George; Kong, David
      Nature Biotechnology (2022), 40 (12), 1874-1879CODEN: NABIF9; ISSN:1087-0156. (Nature Portfolio)
      A pilot program for synthetic biol. education via a scalable distributed network model of distance-based lab. learning can be accessible globally across disciplines and backgrounds.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjtFSlsLzF&md5=5a8f9f979f93a31e57c86f0bca9f1a05