ACS Publications. Most Trusted. Most Cited. Most Read
3D Printing for the Fabrication of Biofilm-Based Functional Living Materials
More

OR SEARCH CITATIONS

My Activity
Publications

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:ACS Synth. Biol. 2019, 8, 7, 1564-1567
  • Free to Read
Viewpoint

3D Printing for the Fabrication of Biofilm-Based Functional Living Materials
Click to copy article linkArticle link copied!

Open PDF

ACS Synthetic Biology

Cite this: ACS Synth. Biol. 2019, 8, 7, 1564–1567
Click to copy citationCitation copied!
https://doi.org/10.1021/acssynbio.9b00192
Published July 19, 2019

Copyright © 2019 American Chemical Society. This publication is available under these Terms of Use.

Request reuse permissions

Abstract

Click to copy section linkSection link copied!
High Resolution Image
Download MS PowerPoint Slide

Bacterial biofilms are three-dimensional networks of cells entangled in a self-generated extracellular polymeric matrix composed of proteins, lipids, polysaccharides, and nucleic acids. Biofilms can establish themselves on virtually any accessible surface and lead to varying impacts ranging from infectious diseases to degradation of toxic chemicals. Biofilms exhibit high mechanical stiffness and are inherently tolerant to adverse conditions including the presence of antibiotics, pollutants, detergents, high temperature, changes in pH, etc. These features make biofilms resilient, which is beneficial for applications in dynamic environments such as bioleaching, bioremediation, materials production, and wastewater purification. We have recently described an easy and cost-effective method for 3D printing of bacteria and have extended this technology for 3D printing of genetically engineered Escherichia coli biofilms. Our 3D printing platform exploits simple alginate chemistry for printing of a bacteria-alginate bioink mixture onto calcium-containing agar surfaces, resulting in the formation of bacteria-encapsulating hydrogels with varying geometries. Bacteria in these hydrogels remain intact, spatially patterned, and viable for several days. Printing of engineered bacteria to produce inducible biofilms leads to formation of multilayered three-dimensional structures that can tolerate harsh chemical treatments. Synthetic biology and material science approaches provide the opportunity to append a wide range of useful functionalities to these 3D-printed biofilms. In this article, we describe the wide range of future applications possible for applying functional 3D-printed biofilms to the construction of living biofilm-derived materials in a large-scale and environmentally stable manner.

This publication is licensed for personal use by The American Chemical Society.

Copyright © 2019 American Chemical Society

Subjects

what are subjects

Keywords

what are keywords
Bacterial biofilms are organic platforms for sustainable nano- or biomaterials production and processing. The matrix components of naturally occurring biofilms are resilient to extreme conditions and demonstrate self-assembly and spatial patterning. (3−5) These features explain why biofilms have recently become hotspots in emerging materials fabrication and additive manufacturing technologies. Biofilm-derived materials have been applied to a diverse range of applications from detoxification of chemicals to personalized human medicine. By using tools of synthetic biology, it is now possible to improve existing functionalities or even add new functions to biofilm-forming bacteria. Such engineered biofilms are constructed by creating genetic fusions in which desired heterologous functional peptides are appended onto biofilm matrix proteins. These chimeric proteins are then actively secreted by the engineered bacteria and self-assemble in the extracellular matrix of the biofilms. (6,7) Synthetic biofilms can exhibit new functionalities deriving from the added peptides while simultaneously retaining their natural functionalities such as resilience, long-term viability, and self-regeneration. (8) Genetically tractable bacteria such as Escherichia coli and Bacillus subtilis have been successfully employed for the creation of synthetic biofilms and engineered materials. (6,8) During the creation of synthetic biofilms, various factors must be evaluated, including the determination of optimal peptide fusion sites, the tolerance of the fusion protein to mutations, the toxicity of the new peptide tags to the bacterial cells, and appropriate functional assays for characterization of the novel biofilm functionalities. The resultant biofilm-derived materials can exhibit marked advantages over materials fabricated by planktonic bacteria cultures, in terms of their resistance to extreme and unexpected environments, reusability, spatial multiscale patterning, and tunable properties.
Fabrication of biofilm-derived functional materials has been further developed with the aid of 3D printing technology. We have recently demonstrated the repurposing of commercial do-it-yourself 3D printers or construction toys to print bacteria via straightforward alginate chemistry. (1,2) Our simple, scalable, and inexpensive approach was used to print biofilms with submillimeter precision that can mimic the spatial heterogeneity of natural biofilms. The spatial resolution of the 3D-printed biofilms is determined by multiple factors including the bioink composition, the concentration of chemicals that induce expression of the modified biofilm proteins, the rheological properties of the bioink, the biocompatibility of the ink with the printed bacteria, the surface smoothness of the printing substrate, etc. 3D printing of bacteria has also been successfully achieved using bioink compositions including gelatin, agarose, hyaluronic acid, fumed silica, and κ-carrageenan. (9,10)
Previously, one major challenge of 3D bioprinting technology was the operating cost. We have addressed this problem by keeping the cost of our customized 3D bioprinters to approximately $350 US dollars. (1,2) Additionally, some inexpensive commercially available 3D printers can perform multichannel printing, which can mix several input components, and should in principle be able to be repurposed to print bacteria. As a first example, it has been recently shown that 3D printing of bacterial spores with good resolution can be achieved with a customized multichannel printing system, operating at higher temperatures. (10) While this printer costs several times more than our 3D bioprinters, its multichannel printing capability provides the option to keep the bacterial cells separated from the bioink scaffold components under different optimal conditions until printing. We expect that creation or repurposing of cost-effective 3D printers that can perform multichannel printing without heating the samples would be ideal for 3D printing of bacteria and engineered biofilms with extended usage applications.
The combination of bacterial 3D printing technology with biofilm biology is a fascinating approach toward translation of these biofilm-derived materials into useful applications. In the following section, we describe the possible applications arising from the combination of these fields (Figure 1).

Figure 1

Figure 1. Possible applications of 3D-printed synthetic biofilms. Bacteria can be genetically engineered to produce structural biofilm proteins (in blue) decorated with specific functional peptides (in green) via heterologous expression in a bacterial strain that has a genetic deletion for structural biofilm proteins. By combining these engineered bacteria with 3D bioprinting, 3D-printed engineered biofilms can be created with multiple potential applications, including (A) Environmental detoxification and bioremediation, (B) Biomedical applications, (C) Tunable materials production with improved mechanical and/or conductive properties, (D) Fabrication of responsive materials, (E) Biocatalysis-driven materials processing, (F) Addressing fundamental research questions, and (G) Creation of reproducible model biofilm systems for studying the structure–function relationships of bacterial biofilms.

High Resolution Image
Download MS PowerPoint Slide

Materials Production and Processing

Click to copy section linkSection link copied!
Given the wide repertoire of natural and artificial biopolymers, diverse synthetic biofilms could be 3D-printed for the creation of bacterially inspired materials with tunable multiscale patterning. (7,11,12) For instance, bacteria in 3D-printed synthetic biofilms could aid in the production of biopolymers such as cellulose, curdlan, and other materials with improved mechanical or electrically conductive properties with interesting biomedical and biotechnological applications. (9)
3D-printed biofilms functionalized with synthetic enzymes can aid in the processing of materials even under conditions of adverse pH, temperature, or exposure to organic solvents. The desired biocatalytic transformation occurs due to the enzymes that are irreversibly immobilized in the extracellular matrix of these biofilms. The enhanced mass transfer rates and surface area in these biofilms results in increased enzymatic activities. Such biofilms could also be engineered to produce scaffolded chemical pathways, in which successive chemical reactions are catalyzed by individual stacked layers of bacteria, leading to production of a single product or a series of products via a relay of reactions. As one example, the printed bacteria could be genetically manipulated to perform complex logic gate functions, (13) such that the output of one layer could serve as the input to the adjacent layer. (14) These sequential reactions would proceed more efficiently in 3D-printed biofilms due to the free diffusion of molecules between the stacked layers and their minimal separation distance, thus leading to multistep transformations. Alternatively, templated assembly of nanoparticles on engineered biofilms could also be used to catalyze multistep hybrid reaction systems. (6,8)
Nonengineered beneficial bacterial biofilms could be 3D-printed as an antifouling coating on building or marine vessel surfaces. These living functional bacteria would use up the oxygen on the surface and in turn could produce compounds that are anticorrosive, thereby preventing corrosion and biofouling. Similarly, probiotic biofilms could be 3D-printed onto various biomedical implant surfaces to prevent device-associated infections caused by pathogenic bacteria. However, the real-time application of such approaches is far from the current realizations and demands further research.

Environmental Detoxification

3D-printed engineered biofilms could be deployed for environmental detoxification purposes including bioremediation, abstraction of rare earth elements (REEs) and heavy metals, removal of assimilable organic carbon, and in wastewater treatment plants. (9,15) Bringing together the higher metabolic potential and specific catabolic nature of active bacteria with the increased surface area and chemical resilience of the biofilm matrix would enable patterned, engineered biofilms to act as a sink capable of absorption and degradation of chemicals from processing liquid streams. Synthetic biofilms displaying selected catabolic enzymes, heavy metal binding proteins, inorganic nanoparticles, or REE-binding domains could be 3D-printed onto filters or onto pipes and reactors in treatment plants to carry out the desired degradation or abstraction activities as the contaminating streams flow past. Analytical techniques such as HPLC–MS or ICP-MS could be used to quantify the amount of chemicals absorbed onto the biofilm matrix components, and the bound residues could then be desorbed with simple acidic or alkaline washes. Metal-binding domains could be additionally added to these synthetic biofilms to facilitate their strong surface attachment such that they could resist detachment forces and withstand multiple sorption–desorption cycles. With appropriate tuning of the bioink porosity, such 3D-printed biofilms could be recyclable and reusable with minimum loss of efficiency. Incorporation of feedback-regulated genetic circuits could be used in situations involving continuous detoxification such that synthetic biofilms are produced only when the specific target chemical is sensed, thereby improving the overall absorption efficiencies.

Fundamental Research

3D printing could be employed to solve fundamental research questions such as understanding the unknown interactions between bacteria species in mixed biofilms or between bacterial biofilms with their eukaryotic hosts. These experiments could be performed by (a) incorporating different bacteria in the same bioink, (b) printing different bacterial bioinks adjacent to each other with shared interfaces, and/or (c) printing layers of host cells overtop of existing mature 3D-printed biofilms or vice versa. Following appropriate exposure times, imaging techniques and -omics approaches (transcriptomics, proteomics, or metabolomics) could then be used on both the bacterial and host samples to decipher their communication and community behavior. Studying these interactions would greatly help in infectious disease management and discovery of new antibiofilm drugs.

Development of Biofilm Model Systems

In natural biofilms, factors like the density of the bacteria and the extracellular matrix components, the distribution of nutrients and signaling molecules, the locations of water channels, and the distribution of molecular oxygen are dynamic variables. The consequences of these variables on the emergent biological (metabolic heterogeneity and antibiotic resistance) and mechanical (cohesiveness, viscoelasticity, resistance to hydrodynamic shear and desiccation) phenotypes in biofilms are not well characterized. 3D printing could be informative in this regard to identify the design principles of biofilms by introducing individual variations in the 3D spatial distribution of biofilm constituents and studying their resultant attributes of biological and mechanical endurance. These studies could lead to development of an engineered and reproducible biofilm model system that mimics the robustness of natural biofilms while maintaining their structure–function relationships over time. Such model biofilms could then be used for practical applications such as testing potential antibiofilm treatments, evaluating the adequacy of mathematical models of biofilms, etc.

Conclusions and Outlook

Click to copy section linkSection link copied!
3D-printed biofilm-derived materials can exhibit defined spatial patterning with improved resolution and attractive functionalities. However, factors such as reusability, scalability, and potential environmental impacts must be closely investigated for individual applications. For instance, the release of genetically modified bacteria from 3D-printed devices could pose a risk to the environment or to human health, and bacterial contamination must be prevented. For societal applications such as drinking water plants, contamination risks could be eliminated by 3D printing cell-free functional extracellular matrix components that were isolated from biofilms by vacuum filtration. Such components will have longer stability and reusability compared to living bacteria and would not need constant maintenance. An interesting potential application could involve 3D printing multifunctional biofilms that can be used in dynamic settings. Such biofilms could be created by 3D printing either a bioink containing a cocktail of multiple genetically engineered bacteria possessing genetic fusions of different functional proteins and biofilm proteins, or layers of such bacteria one over the other. In either case, cross-seeding of engineered biofilm proteins could occur, leading to a combination of different functionalities in the resultant multifunctional biofilms. Another possible application of 3D-printed biofilms is the creation of responsive materials that could alter their chemical or mechanical properties based on specific environmental cues and triggers. The adaptive nature of such materials would impart them with enhanced lifetimes and continuous functionalities.
Overall, the effectiveness, stability, and versatility of 3D bioprinting approaches in combination with the distinct characteristics of bacterial biofilms offer an ideal platform for the fabrication of biofilm-derived products in materials processing and manufacturing.

Author Information

Click to copy section linkSection link copied!
  • Corresponding Author
  • Authors
    • Srikkanth Balasubramanian,  Department of Bionanoscience & Kavli Institute of Nanoscience, Delft University of Technology, 2628 CD Delft, The Netherlands
    • Marie-Eve Aubin-Tam,  Department of Bionanoscience & Kavli Institute of Nanoscience, Delft University of Technology, 2628 CD Delft, The Netherlands,  Orcidhttp://orcid.org/0000-0001-9995-2623
  • Author Contributions

    S.B. and A.S.M. developed the scope of the manuscript. S.B. conducted the literature search and prepared the first draft and the figures. A.S.M. and M.-E.A.-T. assisted in writing of the manuscript and critically reviewed the manuscript. All authors subsequently modified the manuscript jointly. The final manuscript was approved by all the authors.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

Click to copy section linkSection link copied!

This work was supported by the Air Force Office of Scientific Research, Asian Office of Aerospace Research and Development (grant number FA2386-18-1-4059), and by the University of Rochester Department of Biology.

References

Click to copy section linkSection link copied!

This article references 15 other publications.

  1. 1
    Lehner, B. A. E., Schmieden, D. T., and Meyer, A. S. (2017) A straightforward approach for 3D bacterial printing. ACS Synth. Biol. 6, 1124– 1130,  DOI: 10.1021/acssynbio.6b00395
    Google Scholar
    1
    A Straightforward Approach for 3D Bacterial Printing
    Lehner, Benjamin A. E.; Schmieden, Dominik T.; Meyer, Anne S.
    ACS Synthetic Biology (2017), 6 (7), 1124-1130CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Sustainable and personally tailored materials prodn. is an emerging challenge to society. Living organisms can produce and pattern an extraordinarily wide range of different mols. in a sustainable way. These natural systems offer an abundant source of inspiration for the development of new environmentally friendly materials prodn. techniques. In this paper, the authors describe the first steps toward the 3-dimensional printing of bacterial cultures for materials prodn. and patterning. This methodol. combines the capability of bacteria to form new materials with the reproducibility and tailored approach of 3D printing systems. For this purpose, a com. 3D printer was modified for bacterial systems, and new alginate-based bioink chem. was developed. Printing temp., printhead speed, and bioink extrusion rate were all adapted and customized to maximize bacterial health and spatial resoln. of printed structures. The combination of 3D printing technol. with biol. systems enables a sustainable approach for the prodn. of numerous new materials.
  2. 2
    Schmieden, D. T., Basalo Vazquez, S. J., Sanguesa, H., van der Does, M., Idema, T., and Meyer, A. S. (2018) Printing of patterned, engineered E. coli biofilms with a low-cost 3D printer. ACS Synth. Biol. 7, 1328– 1337,  DOI: 10.1021/acssynbio.7b00424
    Google Scholar
    2
    Printing of patterned, engineered E. coli biofilms with a low-cost 3D printer
    Schmieden, Dominik T.; Basalo Vazquez, Samantha J.; Sangueesa, Hector; van der Does, Marit; Idema, Timon; Meyer, Anne S.
    ACS Synthetic Biology (2018), 7 (5), 1328-1337CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Biofilms can grow on virtually any surface available, with impacts ranging from endangering the lives of patients to degrading unwanted water contaminants. Biofilm research is challenging due to the high degree of biofilm heterogeneity. A method for the prodn. of standardized, reproducible, and patterned biofilm-inspired materials could be a boon for biofilm research and allow for completely new engineering applications. Here, we present such a method, combining 3D printing with genetic engineering. We prototyped a low-cost 3D printer that prints bioink, a suspension of bacteria in a soln. of alginate that solidifies on a calcium-contg. substrate. We 3D-printed Escherichia coli in different shapes and in discrete layers, after which the cells survived in the printing matrix for at least 1 wk. When printed bacteria were induced to form curli fibers, the major proteinaceous extracellular component of E. coli biofilms, they remained adherent to the printing substrate and stably spatially patterned even after treatment with a matrix-dissolving agent, indicating that a biofilm-mimicking structure had formed. This work is the first demonstration of patterned, biofilm-inspired living materials that are produced by genetic control over curli formation in combination with spatial control by 3D printing. These materials could be used as living, functional materials in applications such as water filtration, metal ion sequestration, or civil engineering, and potentially as standardizable models for certain curli-contg. biofilms.
  3. 3
    Bjarnsholt, T., Buhlin, K., Dufrene, Y. F., Gomelsky, M., Moroni, A., Ramstedt, M., Rumbaugh, K. P., Schulte, T., Sun, L., Akerlund, B., and Romling, U. (2018) Biofilm formation - what we can learn from recent developments. J. Intern. Med. 284, 332– 345,  DOI: 10.1111/joim.12782
    Google Scholar
    3
    Biofilm formation - what we can learn from recent developments
    Bjarnsholt T; Bjarnsholt T; Buhlin K; Dufrene Y F; Gomelsky M; Moroni A; Ramstedt M; Rumbaugh K P; Schulte T; Schulte T; Sun L; Romling U; ¡ÑÜAkerlund B
    Journal of internal medicine (2018), 284 (4), 332-345 ISSN:.
    Although biofilms have been observed early in the history of microbial research, their impact has only recently been fully recognized. Biofilm infections, which contribute to up to 80% of human microbial infections, are associated with common human disorders, such as diabetes mellitus and poor dental hygiene, but also with medical implants. The associated chronic infections such as wound infections, dental caries and periodontitis significantly enhance morbidity, affect quality of life and can aid development of follow-up diseases such as cancer. Biofilm infections remain challenging to treat and antibiotic monotherapy is often insufficient, although some rediscovered traditional compounds have shown surprising efficiency. Innovative anti-biofilm strategies include application of anti-biofilm small molecules, intrinsic or external stimulation of production of reactive molecules, utilization of materials with antimicrobial properties and dispersion of biofilms by digestion of the extracellular matrix, also in combination with physical biofilm breakdown. Although basic principles of biofilm formation have been deciphered, the molecular understanding of the formation and structural organization of various types of biofilms has just begun to emerge. Basic studies of biofilm physiology have also resulted in an unexpected discovery of cyclic dinucleotide second messengers that are involved in interkingdom crosstalk via specific mammalian receptors. These findings even open up new venues for exploring novel anti-biofilm strategies.
  4. 4
    Flemming, H. C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S. A., and Kjelleberg, S. (2016) Biofilms: an emergent form of bacterial life. Nat. Rev. Microbiol. 14, 563– 575,  DOI: 10.1038/nrmicro.2016.94
    Google Scholar
    4
    Biofilms: an emergent form of bacterial life
    Flemming, Hans-Curt; Wingender, Jost; Szewzyk, Ulrich; Steinberg, Peter; Rice, Scott A.; Kjelleberg, Staffan
    Nature Reviews Microbiology (2016), 14 (9), 563-575CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
    A review. Bacterial biofilms are formed by communities that are embedded in a self-produced matrix of extracellular polymeric substances (EPS). Importantly, bacteria in biofilms exhibit a set of 'emergent properties' that differ substantially from free-living bacterial cells. In this Review, we consider the fundamental role of the biofilm matrix in establishing the emergent properties of biofilms, describing how the characteristic features of biofilms - such as social cooperation, resource capture and enhanced survival of exposure to antimicrobials - all rely on the structural and functional properties of the matrix. Finally, we highlight the value of an ecol. perspective in the study of the emergent properties of biofilms, which enables an appreciation of the ecol. success of biofilms as habitat formers and, more generally, as a bacterial lifestyle.
  5. 5
    Felz, S., Vermeulen, P., van Loosdrecht, M. C. M., and Lin, Y. M. (2019) Chemical characterization methods for the analysis of structural extracellular polymeric substances (EPS). Water Res. 157, 201– 208,  DOI: 10.1016/j.watres.2019.03.068
    Google Scholar
    5
    Chemical characterization methods for the analysis of structural extracellular polymeric substances (EPS)
    Felz, Simon; Vermeulen, Pascalle; van Loosdrecht, Mark C. M.; Lin, Yue Mei
    Water Research (2019), 157 (), 201-208CODEN: WATRAG; ISSN:0043-1354. (Elsevier Ltd.)
    Biofilm structure and functionality depend on extracellular polymeric substances (EPS), but anal. methods for EPS often lack specificity which limits progress of biofilm research. EPS were extd. from aerobic granular sludge and analyzed with frequently applied colorimetric methods. The colorimetric methods were evaluated based on their applicability for EPS anal. EPS fractions of interest were proteins, sugars, uronic acids and phenolic compds. The applied methods (Lowry method, bicinchoninic acid assay, phenol sulfuric acid method, carbazole sulfuric acid method) were investigated in terms of their sensitivity towards the selected std. compd. Interference of compds. present in EPS with the colorimetric methods was further evaluated. All methods showed to be highly depending on the choice of std. compd. and susceptible towards interference by compds. present in EPS. This study shows that currently used colorimetric methods are not capable of accurately characterizing EPS. More advanced methods are needed to be able to draw conclusions about biofilm compn., structure and functionality.
  6. 6
    Nguyen, P. Q., Botyanszki, Z., Tay, P. K., and Joshi, N. S. (2014) Programmable biofilm-based materials from engineered curli nanofibres. Nat. Commun. 5, 4945,  DOI: 10.1038/ncomms5945
    Google Scholar
    6
    Programmable biofilm-based materials from engineered curli nanofibres
    Nguyen, Peter Q.; Botyanszki, Zsofia; Tay, Pei Kun R.; Joshi, Neel S.
    Nature Communications (2014), 5 (), 4945CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    The significant role of biofilms in pathogenicity has spurred research into preventing their formation and promoting their disruption, resulting in overlooked opportunities to develop biofilms as a synthetic biol. platform for self-assembling functional materials. Here we present Biofilm-Integrated Nanofiber Display (BIND) as a strategy for the mol. programming of the bacterial extracellular matrix material by genetically appending peptide domains to the amyloid protein CsgA, the dominant proteinaceous component in Escherichia coli biofilms. These engineered CsgA fusion proteins are successfully secreted and extracellularly self-assemble into amyloid nanofibre networks that retain the functions of the displayed peptide domains. We show the use of BIND to confer diverse artificial functions to the biofilm matrix, such as nanoparticle biotemplating, substrate adhesion, covalent immobilization of proteins or a combination thereof. BIND is a versatile nanobiotechnol. platform for developing robust materials with programmable functions, demonstrating the potential of utilizing biofilms as large-scale designable biomaterials.
  7. 7
    Chen, A. Y., Deng, Z., Billings, A. N., Seker, U. O., Lu, M. Y., Citorik, R. J., Zakeri, B., and Lu, T. K. (2014) Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater. 13, 515– 523,  DOI: 10.1038/nmat3912
    Google Scholar
    7
    Synthesis and patterning of tunable multiscale materials with engineered cells
    Chen, Allen Y.; Deng, Zhengtao; Billings, Amanda N.; Seker, Urartu O. S.; Lu, Michelle Y.; Citorik, Robert J.; Zakeri, Bijan; Lu, Timothy K.
    Nature Materials (2014), 13 (5), 515-523CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)
    Many natural biol. systems-such as biofilms, shells and skeletal tissues-are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid prodn., we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorg. materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based elec. switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells.
  8. 8
    Huang, J., Liu, S., Zhang, C., Wang, X., Pu, J., Ba, F., Xue, S., Ye, H., Zhao, T., Li, K., Wang, Y., Zhang, J., Wang, L., Fan, C., Lu, T. K., and Zhong, C. (2019) Programmable and printable B. subtilis biofilms as engineered living materials. Nat. Chem. Biol. 15, 34– 41,  DOI: 10.1038/s41589-018-0169-2
    Google Scholar
    8
    Programmable and printable Bacillus subtilis biofilms as engineered living materials
    Huang, Jiaofang; Liu, Suying; Zhang, Chen; Wang, Xinyu; Pu, Jiahua; Ba, Fang; Xue, Shuai; Ye, Haifeng; Zhao, Tianxin; Li, Ke; Wang, Yanyi; Zhang, Jicong; Wang, Lihua; Fan, Chunhai; Lu, Timothy K.; Zhong, Chao
    Nature Chemical Biology (2019), 15 (1), 34-41CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
    Bacterial biofilms can be programmed to produce living materials with self-healing and evolvable functionalities. However, the wider use of artificial biofilms has been hindered by limitations on processability and functional protein secretion capacity. We describe a highly flexible and tunable living functional materials platform based on the TasA amyloid machinery of the bacterium Bacillus subtilis. We demonstrate that genetically programmable TasA fusion proteins harboring diverse functional proteins or domains can be secreted and can assemble into diverse extracellular nano-architectures with tunable physicochem. properties. Our engineered biofilms have the viscoelastic behaviors of hydrogels and can be precisely fabricated into microstructures having a diversity of three-dimensional (3D) shapes using 3D printing and microencapsulation techniques. Notably, these long-lasting and environmentally responsive fabricated living materials remain alive, self-regenerative, and functional. This new tunable platform offers previously unattainable properties for a variety of living functional materials having potential applications in biomaterials, biotechnol., and biomedicine.
  9. 9
    Schaffner, M., Ruhs, P. A., Coulter, F., Kilcher, S., and Studart, A. R. (2017) 3D printing of bacteria into functional complex materials. Sci. Adv. 3, eaao6804  DOI: 10.1126/sciadv.aao6804
    Google Scholar
    There is no corresponding record for this reference.
  10. 10
    Gonzalez, L. M., and Voigt, C. A. (2019) Resilient living materials built by printing bacterial spores. bioRxiv 537571.
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Nguyen, P. Q. (2017) Synthetic biology engineering of biofilms as nanomaterials factories. Biochem. Soc. Trans. 45, 585– 597,  DOI: 10.1042/BST20160348
    Google Scholar
    11
    Synthetic biology engineering of biofilms as nanomaterials factories
    Nguyen, Peter Q.
    Biochemical Society Transactions (2017), 45 (3), 585-597CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
    Bottom-up fabrication of nanoscale materials has been a significant focus in materials science for expanding our technol. frontiers. This assembly concept, however, is old news to biol. - all living organisms fabricate themselves using bottom-up principles through a vast self-organizing system of incredibly complex biomols., a marvelous dynamic that we are still attempting to unravel. Can we use what we have gleaned from biol. thus far to illuminate alternative strategies for designer nanomaterial manufg. In the present review article, new synthetic biol. efforts toward using bacterial biofilms as platforms for the synthesis and secretion of programmable nanomaterials are described. Particular focus is given to self-assembling functional amyloids found in bacterial biofilms as re-engineerable modular nanomol. components. Potential applications and existing challenges for this technol. are also explored. This novel approach for repurposing biofilm systems will enable future technologies for using engineered living systems to grow artificial nanomaterials.
  12. 12
    Chen, A. Y., Zhong, C., and Lu, T. K. (2015) Engineering living functional materials. ACS Synth. Biol. 4, 8– 11,  DOI: 10.1021/sb500113b
    Google Scholar
    12
    Engineering Living Functional Materials
    Chen, Allen Y.; Zhong, Chao; Lu, Timothy K.
    ACS Synthetic Biology (2015), 4 (1), 8-11CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    A review. Natural materials, such as bone, integrate living cells composed of org. mols. together with inorg. components. This enables combinations of functionalities, such as mech. strength and the ability to regenerate and remodel, which are not present in existing synthetic materials. Taking a cue from nature, we propose that engineered 'living functional materials' and 'living materials synthesis platforms' that incorporate both living systems and inorg. components could transform the performance and the manufg. of materials. As a proof-of-concept, we recently demonstrated that synthetic gene circuits in Escherichia coli enabled biofilms to be both a functional material in its own right and a materials-synthesis platform. To demonstrate the former, we engineered E. coli biofilms into a chem.-inducer-responsive elec. switch. To demonstrate the latter, we engineered E. coli biofilms to dynamically organize biotic-abiotic materials across multiple length scales, template gold nanorods, gold nanowires, and metal/semiconductor heterostructures, and synthesize semiconductor nanoparticles. Thus, tools from synthetic biol., such as those for artificial gene regulation, can be used to engineer the spatiotemporal characteristics of living systems and to interface living systems with inorg. materials. Such hybrids can possess novel properties enabled by living cells while retaining desirable functionalities of inorg. systems. These systems, as living functional materials and as living materials foundries, would provide a radically different paradigm of materials performance and synthesis-materials possessing multifunctional, self-healing, adaptable, and evolvable properties that are created and organized in a distributed, bottom-up, autonomously assembled, and environmentally sustainable manner.
  13. 13
    Tamsir, A., Tabor, J. J., and Voigt, C. A. (2011) Robust multicellular computing using genetically encoded NOR gates and chemical ’wires. Nature 469, 212– 215,  DOI: 10.1038/nature09565
    Google Scholar
    13
    Robust multicellular computing using genetically encoded NOR gates and chemical "wires'
    Tamsir, Alvin; Tabor, Jeffrey J.; Voigt, Christopher A.
    Nature (London, United Kingdom) (2011), 469 (7329), 212-215CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Computation underlies the organization of cells into higher-order structures, for example during development or the spatial assocn. of bacteria in a biofilm. Each cell performs a simple computational operation, but when combined with cell-cell communication, intricate patterns emerge. Here the authors study this process by combining a simple genetic circuit with quorum sensing to produce more complex computations in space. They construct a simple NOR logic gate in Escherichia coli by arranging two tandem promoters that function as inputs to drive the transcription of a repressor. The repressor inactivates a promoter that serves as the output. Individual colonies of E. coli carry the same NOR gate, but the inputs and outputs are wired to different orthogonal quorum-sensing 'sender' and 'receiver' devices. The quorum mols. form the wires between gates. By arranging the colonies in different spatial configurations, all possible two-input gates are produced, including the difficult XOR and EQUALS functions. The response is strong and robust, with 5- to >300-fold changes between the 'on' and 'off' states. This work helps elucidate the design rules by which simple logic can be harnessed to produce diverse and complex calcns. by rewiring communication between cells.
  14. 14
    Osmekhina, E., Jonkergouw, C., Schmidt, G., Jahangiri, F., Jokinen, V., Franssila, S., and Linder, M. B. (2018) Controlled communication between physically separated bacterial populations in a microfluidic device. Commun. Biol. 1, 97,  DOI: 10.1038/s42003-018-0102-y
    Google Scholar
    14
    Controlled communication between physically separated bacterial populations in a microfluidic device
    Osmekhina Ekaterina; Jonkergouw Christopher; Schmidt Georg; Linder Markus B; Jahangiri Farzin; Jokinen Ville; Franssila Sami
    Communications biology (2018), 1 (), 97 ISSN:.
    The engineering of microbial systems increasingly strives to achieve a co-existence and co-functioning of different populations. By creating interactions, one can utilize combinations of cells where each population has a specialized function, such as regulation or sharing of metabolic burden. Here we describe a microfluidic system that enables long-term and independent growth of fixed and distinctly separate microbial populations, while allowing communication through a thin nano-cellulose filter. Using quorum-sensing signaling, we can couple the populations and show that this leads to a rapid and stable connection over long periods of time. We continue to show that this control over communication can be utilized to drive nonlinear responses. The coupling of separate populations, standardized interaction, and context-independent function lay the foundation for the construction of increasingly complex community-wide dynamic genetic regulatory mechanisms.
  15. 15
    Tay, P. K. R., Basavanna, A. M., and Joshi, N. S. (2018) Repurposing bacterial extracellular matrix for selective and differential abstraction of rare earth elements. Green Chem. 20, 3512,  DOI: 10.1039/C8GC01355A
    Google Scholar
    15
    Re-purposing bacterial extracellular matrix for selective and differential abstraction of rare earth elements
    Tay, Pei Kun R.; Manjula-Basavanna, Avinash; Joshi, Neel S.
    Green Chemistry (2018), 20 (15), 3512-3520CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
    The rare earth elements (REEs) play crit. roles in modern consumer electronics and clean technologies, but unpredictable supply and environmentally unsustainable extn. practices have spurred efforts to develop green methods of recovering the metals from waste streams. In this regard, the authors have re-purposed a bacterial extracellular matrix (ECM) for selective and differential abstraction of REEs. Herein, the curli amyloid fibers in E. coli biofilms are genetically modified to display lanthanide binding tags (LBTs). The curli-LBT filters showed lanthanide specificity in the presence of other metals, with a preference for binding several high-value heavy REEs. Bound lanthanides were readily recovered using a dil. acid wash, and the filters could be re-used for multiple cycles of sorption and desorption with minimal loss of efficiency. The authors' engineered biofilm-derived filters provide a rapid, selective and scalable method for REE sepn. that is more robust compared to conventional cell-based sorbents, and this platform could be adapted to recover other precious metals or commodities.

Cited By

Click to copy section linkSection link copied!
Citation Statements
Explore this article's citation statements on scite.ai
powered by  Scite

This article is cited by 82 publications.

  1. Minghui Xiao, Shuyi Lv, Chunlei Zhu. Bacterial Patterning: A Promising Biofabrication Technique. ACS Applied Bio Materials 2024, 7 (12) , 8008-8018. https://doi.org/10.1021/acsabm.4c00056
  2. Chenjing Lu, Yaying Huang, Jian Cui, Junhua Wu, Chunping Jiang, Xiaosong Gu, Yi Cao, Sheng Yin. Toward Practical Applications of Engineered Living Materials with Advanced Fabrication Techniques. ACS Synthetic Biology 2024, 13 (8) , 2295-2312. https://doi.org/10.1021/acssynbio.4c00259
  3. Yanyi Wang, Qian Zhang, Changhao Ge, Bolin An, Chao Zhong. Programmable Bacterial Biofilms as Engineered Living Materials. Accounts of Materials Research 2024, 5 (7) , 797-808. https://doi.org/10.1021/accountsmr.3c00271
  4. Shan Liu, Muxuan Yang, Cade Smarr, Ge Zhang, Hazel Barton, Weinan Xu. Engineered Living Structures with Shape-Morphing Capability Enabled by 4D Printing with Functional Bacteria. ACS Applied Bio Materials 2024, 7 (5) , 3247-3257. https://doi.org/10.1021/acsabm.4c00223
  5. Bolin An, Yanyi Wang, Yuanyuan Huang, Xinyu Wang, Yuzhu Liu, Dongmin Xun, George M. Church, Zhuojun Dai, Xiao Yi, Tzu-Chieh Tang, Chao Zhong. Engineered Living Materials For Sustainability. Chemical Reviews 2023, 123 (5) , 2349-2419. https://doi.org/10.1021/acs.chemrev.2c00512
  6. Adrien Sarlet, Valentin Ruffine, Kerstin G. Blank, Cécile M. Bidan. Influence of Metal Cations on the Viscoelastic Properties of Escherichia coli Biofilms. ACS Omega 2023, 8 (5) , 4667-4676. https://doi.org/10.1021/acsomega.2c06438
  7. Seunghyun Sim, Yue Hui, David A. Tirrell. 3D-Printable Cellular Composites for the Production of Recombinant Proteins. Biomacromolecules 2022, 23 (11) , 4687-4695. https://doi.org/10.1021/acs.biomac.2c00915
  8. Yixin Zhang, Le You, Pu Deng, Xiaocheng Jiang, Huan-Hsuan Hsu. Self-Assembled Biohybrid: A Living Material To Bridge the Functions between Electronics and Multilevel Biological Modules/Systems. ACS Applied Materials & Interfaces 2022, 14 (28) , 32289-32298. https://doi.org/10.1021/acsami.2c05639
  9. Julia Müller, Anna C. Jäkel, Jonathan Richter, Markus Eder, Elisabeth Falgenhauer, Friedrich C. Simmel. Bacterial Growth, Communication, and Guided Chemotaxis in 3D-Bioprinted Hydrogel Environments. ACS Applied Materials & Interfaces 2022, 14 (14) , 15871-15880. https://doi.org/10.1021/acsami.1c20836
  10. Srikkanth Balasubramanian, Kui Yu, Diana Vasquez Cardenas, Marie-Eve Aubin-Tam, Anne S. Meyer. Emergent Biological Endurance Depends on Extracellular Matrix Composition of Three-Dimensionally Printed Escherichia coli Biofilms. ACS Synthetic Biology 2021, 10 (11) , 2997-3008. https://doi.org/10.1021/acssynbio.1c00290
  11. Tobias Butelmann, Hans Priks, Zoel Parent, Trevor G. Johnston, Tarmo Tamm, Alshakim Nelson, Petri-Jaan Lahtvee, Rahul Kumar. Metabolism Control in 3D-Printed Living Materials Improves Fermentation. ACS Applied Bio Materials 2021, 4 (9) , 7195-7203. https://doi.org/10.1021/acsabm.1c00754
  12. Eva Sanchez-Rexach, Trevor G. Johnston, Coralie Jehanno, Haritz Sardon, Alshakim Nelson. Sustainable Materials and Chemical Processes for Additive Manufacturing. Chemistry of Materials 2020, 32 (17) , 7105-7119. https://doi.org/10.1021/acs.chemmater.0c02008
  13. Shuaiqi Guo, Emilien Dubuc, Yahav Rave, Mick Verhagen, Simone A. E. Twisk, Tim van der Hek, Guido J. M. Oerlemans, Maxime C. M. van den Oetelaar, Laura S. van Hazendonk, Mariska Brüls, Bruno V. Eijkens, Pim L. Joostens, Sander R. Keij, Weizhou Xing, Martijn Nijs, Jitske Stalpers, Manoj Sharma, Marieke Gerth, Roy J. E. A. Boonen, Kees Verduin, Maarten Merkx, Ilja K. Voets, Tom F. A. de Greef. Engineered Living Materials Based on Adhesin-Mediated Trapping of Programmable Cells. ACS Synthetic Biology 2020, 9 (3) , 475-485. https://doi.org/10.1021/acssynbio.9b00404
  14. Ethan Viles, Ethan Heyneman, Shuyi Lin, Virginia Montague, Amir Darabi, Lewis M. Cox, Adrienne Phillips, Robin Gerlach, Erika J. Espinosa-Ortiz, Chelsea Heveran. Mycelium as a scaffold for biomineralized engineered living materials. Cell Reports Physical Science 2025, 6 (4) , 102517. https://doi.org/10.1016/j.xcrp.2025.102517
  15. Jiaqi Qiu, Shuo Ma, Xue Qu. Challenges and Innovative Strategies in 3D Printing of Natural Biomolecular Hydrogels. Nano Select 2025, 8 https://doi.org/10.1002/nano.202400149
  16. Jacob Moran, Kevin B. Wood. From Fluctuations and Disorder to Scaling and Control: The Emergence of Resistance in Microbial Communities. Annual Review of Condensed Matter Physics 2025, 16 (1) , 297-318. https://doi.org/10.1146/annurev-conmatphys-042924-110923
  17. Muhammad Bagas Ananda, Maradhana Agung Marsudi, Indra Jaya Budiarso, Akfiny Hasdi Aimon, Ferry Iskandar, Cian Vyas, Glen Cooper, Paulo J.D.S. Bartolo, Arie Wibowo. 3D printed 2D materials for tissue engineering applications. ChemPhysMater 2025, 5 https://doi.org/10.1016/j.chphma.2024.12.004
  18. Jonathan Nyenhuis, Christopher Heuer, Janina Bahnemann. 3D Printing in Biocatalysis and Biosensing: From General Concepts to Practical Applications. Chemistry – An Asian Journal 2024, 19 (24) https://doi.org/10.1002/asia.202400717
  19. Avinash Manjula-Basavanna, Anna M. Duraj-Thatte, Neel S. Joshi. Mechanically Tunable, Compostable, Healable and Scalable Engineered Living Materials. Nature Communications 2024, 15 (1) https://doi.org/10.1038/s41467-024-53052-4
  20. Mark R. Shannon, Bini Zhou, Adam W. Perriman. Leveraging the Power of Enzymes in Engineered Dead and Living Materials. Advanced Functional Materials 2024, 34 (44) https://doi.org/10.1002/adfm.202404522
  21. Chelsea M. Heveran, Robin Gerlach, Christopher J. Hernandez, Kristen Intemann, Anne S. Meyer, Caroline Ajo-Franklin, Marimikel Charrier, Wilfred V. Srubar, Neel Joshi, Alshakim Nelson, Matthew W. Fields. Unlocking the societal potential of engineered living materials. Matter 2024, 7 (9) , 2846-2858. https://doi.org/10.1016/j.matt.2024.07.011
  22. Xiang Qi, Xinning Liu, Yuyi Gu, Peng Liang. Whole-cell biophotovoltaic systems for renewable energy generation: A systematic analysis of existing knowledge. Bioelectrochemistry 2024, 158 , 108695. https://doi.org/10.1016/j.bioelechem.2024.108695
  23. Yi Lu, Marina Mehling, Siqi Huan, Long Bai, Orlando J. Rojas. Biofabrication with microbial cellulose: from bioadaptive designs to living materials. Chemical Society Reviews 2024, 53 (14) , 7363-7391. https://doi.org/10.1039/D3CS00641G
  24. Josha Herzog, Lea Franke, Yingyao Lai, Pablo Gomez Rossi, Janina Sachtleben, Dirk Weuster-Botz. 3D bioprinting of microorganisms: principles and applications. Bioprocess and Biosystems Engineering 2024, 47 (4) , 443-461. https://doi.org/10.1007/s00449-023-02965-3
  25. Oliver Denton, Yifei Wan, Laura Beattie, Téa Jack, Preston McGoldrick, Holly McAllister, Cara Mullan, Catriona M. Douglas, Wenmiao Shu. Understanding the Role of Biofilms in Acute Recurrent Tonsillitis through 3D Bioprinting of a Novel Gelatin-PEGDA Hydrogel. Bioengineering 2024, 11 (3) , 202. https://doi.org/10.3390/bioengineering11030202
  26. Jeong‐Joo Oh, Satya Ammu, Vivian Dorine Vriend, Roland Kieffer, Friedrich Hans Kleiner, Srikkanth Balasubramanian, Elvin Karana, Kunal Masania, Marie‐Eve Aubin‐Tam. Growth, Distribution, and Photosynthesis of Chlamydomonas Reinhardtii in 3D Hydrogels. Advanced Materials 2024, 36 (2) https://doi.org/10.1002/adma.202305505
  27. Seon-Won Yoon, Sang-Yeob Kim, Joon-Seong Jeon, Seungdae Oh, Sang-Yeop Chung, Ji-Su Kim, Sung-Kyu Maeng. 3D-printed Chlorella vulgaris biocarriers: A novel approach to wastewater treatment. Journal of Water Process Engineering 2024, 57 , 104711. https://doi.org/10.1016/j.jwpe.2023.104711
  28. Emily Lazarus, Anne S. Meyer, Kaoru Ikuma, Iris V. Rivero. Three dimensional printed biofilms: Fabrication, design and future biomedical and environmental applications. Microbial Biotechnology 2024, 17 (1) https://doi.org/10.1111/1751-7915.14360
  29. Roberta Rovelli, Beatrice Cecchini, Lorenzo Zavagna, Bahareh Azimi, Claudio Ricci, Semih Esin, Mario Milazzo, Giovanna Batoni, Serena Danti. Emerging Multiscale Biofabrication Approaches for Bacteriotherapy. Molecules 2024, 29 (2) , 533. https://doi.org/10.3390/molecules29020533
  30. Abraham Samuel Finny. 3D bioprinting in bioremediation: a comprehensive review of principles, applications, and future directions. PeerJ 2024, 12 , e16897. https://doi.org/10.7717/peerj.16897
  31. Daniela Peneda Pacheco, Federico Bertoglio, Cosmin Butnarasu, Natalia Suarez Vargas, Giuseppe Guagliano, Anna Ziccarelli, Francesco Briatico‐Vangosa, Vincenzo Ruzzi, Stefano Buzzaccaro, Roberto Piazza, Sebastião van Uden, Elena Crotti, Sonja Visentin, Livia Visai, Paola Petrini. Heterogeneity Governs 3D‐Cultures of Clinically Relevant Microbial Communities. Advanced Functional Materials 2023, 33 (50) https://doi.org/10.1002/adfm.202306116
  32. Claretta J. Sullivan, Kennedy Brown, Chia-Suei Hung, Joseph Kuo-Hsiang Tang, Mark DeSimone, Vincent Chen, Pamela F. Lloyd, Maneesh Gupta, Abby Juhl, Wendy Crookes-Goodson, Milana Vasudev, Patrick B. Dennis, Nancy Kelley-Loughnane. Iridescent biofilms of Cellulophaga lytica are tunable platforms for scalable, ordered materials. Scientific Reports 2023, 13 (1) https://doi.org/10.1038/s41598-023-38797-0
  33. Hanfeng Zhai, Jingjie Yeo. Controlling biofilm transport with porous metamaterials designed with Bayesian learning. Journal of the Mechanical Behavior of Biomedical Materials 2023, 147 , 106127. https://doi.org/10.1016/j.jmbbm.2023.106127
  34. Chelsea M. Heveran, Christopher J. Hernandez. Make engineered living materials carry their weight. Matter 2023, 6 (11) , 3705-3718. https://doi.org/10.1016/j.matt.2023.07.023
  35. Yujia Jiang, Ruofan Wu, Wenming Zhang, Fengxue Xin, Min Jiang. Construction of stable microbial consortia for effective biochemical synthesis. Trends in Biotechnology 2023, 41 (11) , 1430-1441. https://doi.org/10.1016/j.tibtech.2023.05.008
  36. Pankaj Sharma, Vinay Jain. An overview of current advances and pharmaceutical uses of 3D and 4D printing. Exploration of Medicine 2023, , 560-575. https://doi.org/10.37349/emed.2023.00161
  37. Leo Ming Pong Sun, Andrew Ching-Yuet To, . Inexpensive DIY Bioprinting in a Secondary School Setting. Journal of Microbiology & Biology Education 2023, 24 (2) https://doi.org/10.1128/jmbe.00124-22
  38. Michal Walczak, Leonardo Mancini, Jiayi Xu, Federica Raguseo, Jurij Kotar, Pietro Cicuta, Lorenzo Di Michele. A Synthetic Signaling Network Imitating the Action of Immune Cells in Response to Bacterial Metabolism. Advanced Materials 2023, 35 (33) https://doi.org/10.1002/adma.202301562
  39. Srinivas Sulugodu Ramachandra, Patricia Wright, Pingping Han, Abdalla Abdal‐hay, Ryan S. B. Lee, Saso Ivanovski. Evaluating models and assessment techniques for understanding oral biofilm complexity. MicrobiologyOpen 2023, 12 (4) https://doi.org/10.1002/mbo3.1377
  40. Xianyong Li, Rui Kong, Jiankun Wang, Jin Wu, Ketai He, Xiaoling Wang. The formation mechanism of Bacillus subtilis biofilm surface morphology under competitive environment. Canadian Journal of Microbiology 2023, 69 (7) , 251-261. https://doi.org/10.1139/cjm-2023-0014
  41. Sudipto Datta, . Advantage of Alginate Bioinks in Biofabrication for Various Tissue Engineering Applications. International Journal of Polymer Science 2023, 2023 , 1-20. https://doi.org/10.1155/2023/6661452
  42. Hao Gao, Hanxiao Wang, Yiqing Zhang, Yujiao Wang, Guannan Liu, Quanyu Zhao, Ziyi Yu, Fengxue Xin, Wenming Zhang. Design and optimization of artificial light-driven microbial consortia for the sustainable growth and biosynthesis of 2-phenylethanol. Chemical Engineering Journal 2023, 466 , 143050. https://doi.org/10.1016/j.cej.2023.143050
  43. Matthias Portius, Christian Danneberg, Tilo Pompe. Biomaterial approaches for engineering and analyzing structure and metabolic states of microbial consortia within biofilms. Current Opinion in Biotechnology 2023, 81 , 102916. https://doi.org/10.1016/j.copbio.2023.102916
  44. Anthony J. Kyser, Mohammadali Masigol, Mohamed Y. Mahmoud, Mark Ryan, Warren G. Lewis, Amanda L. Lewis, Hermann B. Frieboes, Jill M. Steinbach-Rankins. Fabrication and characterization of bioprints with Lactobacillus crispatus for vaginal application. Journal of Controlled Release 2023, 357 , 545-560. https://doi.org/10.1016/j.jconrel.2023.04.023
  45. Barbara Schamberger, Ricardo Ziege, Karine Anselme, Martine Ben Amar, Michał Bykowski, André P. G. Castro, Amaia Cipitria, Rhoslyn A. Coles, Rumiana Dimova, Michaela Eder, Sebastian Ehrig, Luis M. Escudero, Myfanwy E. Evans, Paulo R. Fernandes, Peter Fratzl, Liesbet Geris, Notburga Gierlinger, Edouard Hannezo, Aleš Iglič, Jacob J. K. Kirkensgaard, Philip Kollmannsberger, Łucja Kowalewska, Nicholas A. Kurniawan, Ioannis Papantoniou, Laurent Pieuchot, Tiago H. V. Pires, Lars D. Renner, Andrew O. Sageman‐Furnas, Gerd E. Schröder‐Turk, Anupam Sengupta, Vikas R. Sharma, Antonio Tagua, Caterina Tomba, Xavier Trepat, Sarah L. Waters, Edwina F. Yeo, Andreas Roschger, Cécile M. Bidan, John W. C. Dunlop. Curvature in Biological Systems: Its Quantification, Emergence, and Implications across the Scales. Advanced Materials 2023, 35 (13) https://doi.org/10.1002/adma.202206110
  46. Yifei Liu, Xiudong Xia, Zhen Liu, Mingsheng Dong. The Next Frontier of 3D Bioprinting: Bioactive Materials Functionalized by Bacteria. Small 2023, 19 (10) https://doi.org/10.1002/smll.202205949
  47. G. Crivello, L. Fracchia, G. Ciardelli, M. Boffito, C. Mattu. In Vitro Models of Bacterial Biofilms: Innovative Tools to Improve Understanding and Treatment of Infections. Nanomaterials 2023, 13 (5) , 904. https://doi.org/10.3390/nano13050904
  48. Robert D. Murphy, Ronnie V. Garcia, Seung J. Oh, Tanner J. Wood, Kyoo D. Jo, Javier Read de Alaniz, Ed Perkins, Craig J. Hawker. Tailored Polypeptide Star Copolymers for 3D Printing of Bacterial Composites Via Direct Ink Writing. Advanced Materials 2023, 35 (3) https://doi.org/10.1002/adma.202207542
  49. Katherine Ansbro, Cher Farrugia, Graham P. Stafford, Joanna Shepherd. Polymicrobial Biofilm Models: The Case of Periodontal Disease as an Example. 2023, 195-230. https://doi.org/10.1007/978-3-031-15349-5_6
  50. Alireza Nouri, Anahita Rohani Shirvan, Yuncang Li, Cuie Wen. Surface modification of additively manufactured metallic biomaterials with active antipathogenic properties. Smart Materials in Manufacturing 2023, 1 , 100001. https://doi.org/10.1016/j.smmf.2022.100001
  51. Sara Molinari, Robert F. Tesoriero, Dong Li, Swetha Sridhar, Rong Cai, Jayashree Soman, Kathleen R. Ryan, Paul D. Ashby, Caroline M. Ajo-Franklin. A de novo matrix for macroscopic living materials from bacteria. Nature Communications 2022, 13 (1) https://doi.org/10.1038/s41467-022-33191-2
  52. Hao Gao, Minhui Li, Lu Yang, Yujia Jiang, Wankui Jiang, Ziyi Yu, Wenming Zhang, Fengxue Xin, Min Jiang. Spatial niche construction of a consortium-based consolidated bioprocessing system. Green Chemistry 2022, 24 (20) , 7941-7950. https://doi.org/10.1039/D2GC01850K
  53. Yajuan Su, Jaime T. Yrastorza, Mitchell Matis, Jenna Cusick, Siwei Zhao, Guangshun Wang, Jingwei Xie. Biofilms: Formation, Research Models, Potential Targets, and Methods for Prevention and Treatment. Advanced Science 2022, 9 (29) https://doi.org/10.1002/advs.202203291
  54. Daniel D. Lewis, Ting Gong, Yuanwei Xu, Cheemeng Tan. Frequency dependent growth of bacteria in living materials. Frontiers in Bioengineering and Biotechnology 2022, 10 https://doi.org/10.3389/fbioe.2022.948483
  55. Yiting Ze, Ruixin Wang, Hanzhi Deng, Zheqing Zhou, Xiaoju Chen, Linyang Huang, Yang Yao. Three-dimensional bioprinting: A cutting-edge tool for designing and fabricating engineered living materials. Biomaterials Advances 2022, 140 , 213053. https://doi.org/10.1016/j.bioadv.2022.213053
  56. Suying Liu, Jiaofang Huang, Chen Zhang, Lihua Wang, Chunhai Fan, Chao Zhong. Probing the growth and mechanical properties of Bacillus subtilis biofilms through genetic mutation strategies. Synthetic and Systems Biotechnology 2022, 7 (3) , 965-971. https://doi.org/10.1016/j.synbio.2022.05.005
  57. Pooja Jain, Himanshu Kathuria, Nileshkumar Dubey. Advances in 3D bioprinting of tissues/organs for regenerative medicine and in-vitro models. Biomaterials 2022, 287 , 121639. https://doi.org/10.1016/j.biomaterials.2022.121639
  58. Ann Chie Lim, Shirley Gee Hoon Tang, Noraziah Mohamad Zin, Abdul Mutalib Maisarah, Indang Ariati Ariffin, Pin Jern Ker, Teuku Meurah Indra Mahlia. Chemical Composition, Antioxidant, Antibacterial, and Antibiofilm Activities of Backhousia citriodora Essential Oil. Molecules 2022, 27 (15) , 4895. https://doi.org/10.3390/molecules27154895
  59. Xinyue Liu, Maria Eugenia Inda, Yong Lai, Timothy K. Lu, Xuanhe Zhao. Engineered Living Hydrogels. Advanced Materials 2022, 34 (26) https://doi.org/10.1002/adma.202201326
  60. Daniel Wangpraseurt, Shangting You, Yazhi Sun, Shaochen Chen. Biomimetic 3D living materials powered by microorganisms. Trends in Biotechnology 2022, 40 (7) , 843-857. https://doi.org/10.1016/j.tibtech.2022.01.003
  61. Raffaele Pugliese, Stefano Regondi. Artificial Intelligence-Empowered 3D and 4D Printing Technologies toward Smarter Biomedical Materials and Approaches. Polymers 2022, 14 (14) , 2794. https://doi.org/10.3390/polym14142794
  62. Kaavian Shariati, Andrea Shin Ling, Stephanie Fuchs, Benjamin Dillenburger, Wanjun Liu, Minglin Ma. Hylozoic by Design: Converging Material and Biological Complexities for Cell‐Driven Living Materials with 4D Behaviors. Advanced Functional Materials 2022, 32 (7) https://doi.org/10.1002/adfm.202108057
  63. Fukun He, Yangteng Ou, Ji Liu, Qiu Huang, Bao Tang, Fengxue Xin, Jing Zhang, Min Jiang, Su Chen, Ziyi Yu. 3D Printed Biocatalytic Living Materials with Dual‐Network Reinforced Bioinks. Small 2022, 18 (6) https://doi.org/10.1002/smll.202104820
  64. Rollin J. Jones, Elizabeth A. Delesky, Sherri M. Cook, Jeffrey C. Cameron, Mija H. Hubler, Wil V. Srubar. Engineered Living Materials for Construction. 2022, 187-216. https://doi.org/10.1007/978-3-030-92949-7_7
  65. Gökçe Özkul, Merve Yavuz, Nedim Hacıosmanoğlu, Büşra Merve Kırpat, Urartu Özgür Şafak Şeker. Design and applications of self-assembled soft living materials using synthetic biology. 2022, 361-372. https://doi.org/10.1016/B978-0-12-824469-2.00006-3
  66. Bin Xu, Zhe Li, Yujia Jiang, Minjiao Chen, Boryann Chen, Fengxue Xin, Weiliang Dong, Min Jiang. Recent advances in the improvement of bi-directional electron transfer between abiotic/biotic interfaces in electron-assisted biosynthesis system. Biotechnology Advances 2022, 54 , 107810. https://doi.org/10.1016/j.biotechadv.2021.107810
  67. Elif N. Hayta, Marvin J. Ertelt, Martin Kretschmer, Oliver Lieleg. Bacterial Materials: Applications of Natural and Modified Biofilms. Advanced Materials Interfaces 2021, 8 (21) https://doi.org/10.1002/admi.202101024
  68. Dhinakaran Veeman, M. Swapna Sai, P. Sureshkumar, T. Jagadeesha, L. Natrayan, M. Ravichandran, Wubishet Degife Mammo, . Additive Manufacturing of Biopolymers for Tissue Engineering and Regenerative Medicine: An Overview, Potential Applications, Advancements, and Trends. International Journal of Polymer Science 2021, 2021 , 1-20. https://doi.org/10.1155/2021/4907027
  69. Adrian Augustyniak, Pawel Sikora, Bartłomiej Grygorcewicz, Daneish Despot, Burga Braun, Rafał Rakoczy, Ulrich Szewzyk, Matthias Barjenbruch, Dietmar Stephan. Biofilms in the gravity sewer interfaces: making a friend from a foe. Reviews in Environmental Science and Bio/Technology 2021, 20 (3) , 795-813. https://doi.org/10.1007/s11157-021-09582-0
  70. Srikkanth Balasubramanian, Kui Yu, Anne S. Meyer, Elvin Karana, Marie‐Eve Aubin‐Tam. Bioprinting of Regenerative Photosynthetic Living Materials. Advanced Functional Materials 2021, 31 (31) https://doi.org/10.1002/adfm.202011162
  71. Wil V. Srubar. Engineered Living Materials: Taxonomies and Emerging Trends. Trends in Biotechnology 2021, 39 (6) , 574-583. https://doi.org/10.1016/j.tibtech.2020.10.009
  72. Priyadarshini Chandrashekhar, Farnaz Minooei, Wenndy Arreguin, Mohammadali Masigol, Jill M. Steinbach-Rankins. Perspectives on Existing and Novel Alternative Intravaginal Probiotic Delivery Methods in the Context of Bacterial Vaginosis Infection. The AAPS Journal 2021, 23 (3) https://doi.org/10.1208/s12248-021-00602-z
  73. Yanshan Yu, Lijuan Wang, Pingjun Ma. Nanocrystalline collagen-based bone materials in skeletal optimization in patients. Nanomaterials and Energy 2021, 10 (1) , 9-16. https://doi.org/10.1680/jnaen.20.00005
  74. Ecem Saygili, Mohamed S. Draz. 3D Printable Gel-Inks for Microbes and Microbial Structures. 2021, 333-353. https://doi.org/10.1007/978-981-16-4667-6_10
  75. Sergey Chapek, Sergey Golovin, Michael Chikindas, Svetlana Ponomareva, Dmitry Rudoy, Anastasiya Olshevskaya, , , . Application of 3D bioprinting in the study of bacterial biofilms. E3S Web of Conferences 2021, 273 , 13010. https://doi.org/10.1051/e3sconf/202127313010
  76. M. R. Sharipova, A. M. Mardanova, N. L. Rudakova, D. S. Pudova. Bistability and Formation of the Biofilm Matrix as Adaptive Mechanisms during the Stationary Phase of Bacillus subtilis. Microbiology 2021, 90 (1) , 20-36. https://doi.org/10.1134/S002626172006017X
  77. Iago Grobas, Dario G. Bazzoli, Munehiro Asally. Biofilm and swarming emergent behaviours controlled through the aid of biophysical understanding and tools. Biochemical Society Transactions 2020, 48 (6) , 2903-2913. https://doi.org/10.1042/BST20200972
  78. Yuan Su, Chuan Liu, Huan Fang, Dawei Zhang. Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine. Microbial Cell Factories 2020, 19 (1) https://doi.org/10.1186/s12934-020-01436-8
  79. Ram Surya Gona, Anne S. Meyer. Engineered proteins and three-dimensional printing of living materials. MRS Bulletin 2020, 45 (12) , 1034-1038. https://doi.org/10.1557/mrs.2020.298
  80. Adam Chyzy, Marta E. Plonska-Brzezinska. Hydrogel Properties and Their Impact on Regenerative Medicine and Tissue Engineering. Molecules 2020, 25 (24) , 5795. https://doi.org/10.3390/molecules25245795
  81. Giuseppe Arrabito, Vittorio Ferrara, Aurelio Bonasera, Bruno Pignataro. Artificial Biosystems by Printing Biology. Small 2020, 16 (27) https://doi.org/10.1002/smll.201907691
  82. Lina M. González, Nikita Mukhitov, Christopher A. Voigt. Resilient living materials built by printing bacterial spores. Nature Chemical Biology 2020, 16 (2) , 126-133. https://doi.org/10.1038/s41589-019-0412-5
Download PDF
Go to ACS Synthetic Biology
Get e-Alerts
Get e-Alerts

ACS Synthetic Biology

Cite this: ACS Synth. Biol. 2019, 8, 7, 1564–1567
Click to copy citationCitation copied!
https://doi.org/10.1021/acssynbio.9b00192
Published July 19, 2019

Copyright © 2019 American Chemical Society. This publication is available under these Terms of Use.

Request reuse permissions

Article Views

12k

Altmetric

-

Citations

82
Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. Possible applications of 3D-printed synthetic biofilms. Bacteria can be genetically engineered to produce structural biofilm proteins (in blue) decorated with specific functional peptides (in green) via heterologous expression in a bacterial strain that has a genetic deletion for structural biofilm proteins. By combining these engineered bacteria with 3D bioprinting, 3D-printed engineered biofilms can be created with multiple potential applications, including (A) Environmental detoxification and bioremediation, (B) Biomedical applications, (C) Tunable materials production with improved mechanical and/or conductive properties, (D) Fabrication of responsive materials, (E) Biocatalysis-driven materials processing, (F) Addressing fundamental research questions, and (G) Creation of reproducible model biofilm systems for studying the structure–function relationships of bacterial biofilms.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 15 other publications.

    1. 1
      Lehner, B. A. E., Schmieden, D. T., and Meyer, A. S. (2017) A straightforward approach for 3D bacterial printing. ACS Synth. Biol. 6, 1124– 1130,  DOI: 10.1021/acssynbio.6b00395
      1
      A Straightforward Approach for 3D Bacterial Printing
      Lehner, Benjamin A. E.; Schmieden, Dominik T.; Meyer, Anne S.
      ACS Synthetic Biology (2017), 6 (7), 1124-1130CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Sustainable and personally tailored materials prodn. is an emerging challenge to society. Living organisms can produce and pattern an extraordinarily wide range of different mols. in a sustainable way. These natural systems offer an abundant source of inspiration for the development of new environmentally friendly materials prodn. techniques. In this paper, the authors describe the first steps toward the 3-dimensional printing of bacterial cultures for materials prodn. and patterning. This methodol. combines the capability of bacteria to form new materials with the reproducibility and tailored approach of 3D printing systems. For this purpose, a com. 3D printer was modified for bacterial systems, and new alginate-based bioink chem. was developed. Printing temp., printhead speed, and bioink extrusion rate were all adapted and customized to maximize bacterial health and spatial resoln. of printed structures. The combination of 3D printing technol. with biol. systems enables a sustainable approach for the prodn. of numerous new materials.
    2. 2
      Schmieden, D. T., Basalo Vazquez, S. J., Sanguesa, H., van der Does, M., Idema, T., and Meyer, A. S. (2018) Printing of patterned, engineered E. coli biofilms with a low-cost 3D printer. ACS Synth. Biol. 7, 1328– 1337,  DOI: 10.1021/acssynbio.7b00424
      2
      Printing of patterned, engineered E. coli biofilms with a low-cost 3D printer
      Schmieden, Dominik T.; Basalo Vazquez, Samantha J.; Sangueesa, Hector; van der Does, Marit; Idema, Timon; Meyer, Anne S.
      ACS Synthetic Biology (2018), 7 (5), 1328-1337CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Biofilms can grow on virtually any surface available, with impacts ranging from endangering the lives of patients to degrading unwanted water contaminants. Biofilm research is challenging due to the high degree of biofilm heterogeneity. A method for the prodn. of standardized, reproducible, and patterned biofilm-inspired materials could be a boon for biofilm research and allow for completely new engineering applications. Here, we present such a method, combining 3D printing with genetic engineering. We prototyped a low-cost 3D printer that prints bioink, a suspension of bacteria in a soln. of alginate that solidifies on a calcium-contg. substrate. We 3D-printed Escherichia coli in different shapes and in discrete layers, after which the cells survived in the printing matrix for at least 1 wk. When printed bacteria were induced to form curli fibers, the major proteinaceous extracellular component of E. coli biofilms, they remained adherent to the printing substrate and stably spatially patterned even after treatment with a matrix-dissolving agent, indicating that a biofilm-mimicking structure had formed. This work is the first demonstration of patterned, biofilm-inspired living materials that are produced by genetic control over curli formation in combination with spatial control by 3D printing. These materials could be used as living, functional materials in applications such as water filtration, metal ion sequestration, or civil engineering, and potentially as standardizable models for certain curli-contg. biofilms.
    3. 3
      Bjarnsholt, T., Buhlin, K., Dufrene, Y. F., Gomelsky, M., Moroni, A., Ramstedt, M., Rumbaugh, K. P., Schulte, T., Sun, L., Akerlund, B., and Romling, U. (2018) Biofilm formation - what we can learn from recent developments. J. Intern. Med. 284, 332– 345,  DOI: 10.1111/joim.12782
      3
      Biofilm formation - what we can learn from recent developments
      Bjarnsholt T; Bjarnsholt T; Buhlin K; Dufrene Y F; Gomelsky M; Moroni A; Ramstedt M; Rumbaugh K P; Schulte T; Schulte T; Sun L; Romling U; ¡ÑÜAkerlund B
      Journal of internal medicine (2018), 284 (4), 332-345 ISSN:.
      Although biofilms have been observed early in the history of microbial research, their impact has only recently been fully recognized. Biofilm infections, which contribute to up to 80% of human microbial infections, are associated with common human disorders, such as diabetes mellitus and poor dental hygiene, but also with medical implants. The associated chronic infections such as wound infections, dental caries and periodontitis significantly enhance morbidity, affect quality of life and can aid development of follow-up diseases such as cancer. Biofilm infections remain challenging to treat and antibiotic monotherapy is often insufficient, although some rediscovered traditional compounds have shown surprising efficiency. Innovative anti-biofilm strategies include application of anti-biofilm small molecules, intrinsic or external stimulation of production of reactive molecules, utilization of materials with antimicrobial properties and dispersion of biofilms by digestion of the extracellular matrix, also in combination with physical biofilm breakdown. Although basic principles of biofilm formation have been deciphered, the molecular understanding of the formation and structural organization of various types of biofilms has just begun to emerge. Basic studies of biofilm physiology have also resulted in an unexpected discovery of cyclic dinucleotide second messengers that are involved in interkingdom crosstalk via specific mammalian receptors. These findings even open up new venues for exploring novel anti-biofilm strategies.
    4. 4
      Flemming, H. C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S. A., and Kjelleberg, S. (2016) Biofilms: an emergent form of bacterial life. Nat. Rev. Microbiol. 14, 563– 575,  DOI: 10.1038/nrmicro.2016.94
      4
      Biofilms: an emergent form of bacterial life
      Flemming, Hans-Curt; Wingender, Jost; Szewzyk, Ulrich; Steinberg, Peter; Rice, Scott A.; Kjelleberg, Staffan
      Nature Reviews Microbiology (2016), 14 (9), 563-575CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
      A review. Bacterial biofilms are formed by communities that are embedded in a self-produced matrix of extracellular polymeric substances (EPS). Importantly, bacteria in biofilms exhibit a set of 'emergent properties' that differ substantially from free-living bacterial cells. In this Review, we consider the fundamental role of the biofilm matrix in establishing the emergent properties of biofilms, describing how the characteristic features of biofilms - such as social cooperation, resource capture and enhanced survival of exposure to antimicrobials - all rely on the structural and functional properties of the matrix. Finally, we highlight the value of an ecol. perspective in the study of the emergent properties of biofilms, which enables an appreciation of the ecol. success of biofilms as habitat formers and, more generally, as a bacterial lifestyle.
    5. 5
      Felz, S., Vermeulen, P., van Loosdrecht, M. C. M., and Lin, Y. M. (2019) Chemical characterization methods for the analysis of structural extracellular polymeric substances (EPS). Water Res. 157, 201– 208,  DOI: 10.1016/j.watres.2019.03.068
      5
      Chemical characterization methods for the analysis of structural extracellular polymeric substances (EPS)
      Felz, Simon; Vermeulen, Pascalle; van Loosdrecht, Mark C. M.; Lin, Yue Mei
      Water Research (2019), 157 (), 201-208CODEN: WATRAG; ISSN:0043-1354. (Elsevier Ltd.)
      Biofilm structure and functionality depend on extracellular polymeric substances (EPS), but anal. methods for EPS often lack specificity which limits progress of biofilm research. EPS were extd. from aerobic granular sludge and analyzed with frequently applied colorimetric methods. The colorimetric methods were evaluated based on their applicability for EPS anal. EPS fractions of interest were proteins, sugars, uronic acids and phenolic compds. The applied methods (Lowry method, bicinchoninic acid assay, phenol sulfuric acid method, carbazole sulfuric acid method) were investigated in terms of their sensitivity towards the selected std. compd. Interference of compds. present in EPS with the colorimetric methods was further evaluated. All methods showed to be highly depending on the choice of std. compd. and susceptible towards interference by compds. present in EPS. This study shows that currently used colorimetric methods are not capable of accurately characterizing EPS. More advanced methods are needed to be able to draw conclusions about biofilm compn., structure and functionality.
    6. 6
      Nguyen, P. Q., Botyanszki, Z., Tay, P. K., and Joshi, N. S. (2014) Programmable biofilm-based materials from engineered curli nanofibres. Nat. Commun. 5, 4945,  DOI: 10.1038/ncomms5945
      6
      Programmable biofilm-based materials from engineered curli nanofibres
      Nguyen, Peter Q.; Botyanszki, Zsofia; Tay, Pei Kun R.; Joshi, Neel S.
      Nature Communications (2014), 5 (), 4945CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      The significant role of biofilms in pathogenicity has spurred research into preventing their formation and promoting their disruption, resulting in overlooked opportunities to develop biofilms as a synthetic biol. platform for self-assembling functional materials. Here we present Biofilm-Integrated Nanofiber Display (BIND) as a strategy for the mol. programming of the bacterial extracellular matrix material by genetically appending peptide domains to the amyloid protein CsgA, the dominant proteinaceous component in Escherichia coli biofilms. These engineered CsgA fusion proteins are successfully secreted and extracellularly self-assemble into amyloid nanofibre networks that retain the functions of the displayed peptide domains. We show the use of BIND to confer diverse artificial functions to the biofilm matrix, such as nanoparticle biotemplating, substrate adhesion, covalent immobilization of proteins or a combination thereof. BIND is a versatile nanobiotechnol. platform for developing robust materials with programmable functions, demonstrating the potential of utilizing biofilms as large-scale designable biomaterials.
    7. 7
      Chen, A. Y., Deng, Z., Billings, A. N., Seker, U. O., Lu, M. Y., Citorik, R. J., Zakeri, B., and Lu, T. K. (2014) Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater. 13, 515– 523,  DOI: 10.1038/nmat3912
      7
      Synthesis and patterning of tunable multiscale materials with engineered cells
      Chen, Allen Y.; Deng, Zhengtao; Billings, Amanda N.; Seker, Urartu O. S.; Lu, Michelle Y.; Citorik, Robert J.; Zakeri, Bijan; Lu, Timothy K.
      Nature Materials (2014), 13 (5), 515-523CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)
      Many natural biol. systems-such as biofilms, shells and skeletal tissues-are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid prodn., we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorg. materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based elec. switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells.
    8. 8
      Huang, J., Liu, S., Zhang, C., Wang, X., Pu, J., Ba, F., Xue, S., Ye, H., Zhao, T., Li, K., Wang, Y., Zhang, J., Wang, L., Fan, C., Lu, T. K., and Zhong, C. (2019) Programmable and printable B. subtilis biofilms as engineered living materials. Nat. Chem. Biol. 15, 34– 41,  DOI: 10.1038/s41589-018-0169-2
      8
      Programmable and printable Bacillus subtilis biofilms as engineered living materials
      Huang, Jiaofang; Liu, Suying; Zhang, Chen; Wang, Xinyu; Pu, Jiahua; Ba, Fang; Xue, Shuai; Ye, Haifeng; Zhao, Tianxin; Li, Ke; Wang, Yanyi; Zhang, Jicong; Wang, Lihua; Fan, Chunhai; Lu, Timothy K.; Zhong, Chao
      Nature Chemical Biology (2019), 15 (1), 34-41CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
      Bacterial biofilms can be programmed to produce living materials with self-healing and evolvable functionalities. However, the wider use of artificial biofilms has been hindered by limitations on processability and functional protein secretion capacity. We describe a highly flexible and tunable living functional materials platform based on the TasA amyloid machinery of the bacterium Bacillus subtilis. We demonstrate that genetically programmable TasA fusion proteins harboring diverse functional proteins or domains can be secreted and can assemble into diverse extracellular nano-architectures with tunable physicochem. properties. Our engineered biofilms have the viscoelastic behaviors of hydrogels and can be precisely fabricated into microstructures having a diversity of three-dimensional (3D) shapes using 3D printing and microencapsulation techniques. Notably, these long-lasting and environmentally responsive fabricated living materials remain alive, self-regenerative, and functional. This new tunable platform offers previously unattainable properties for a variety of living functional materials having potential applications in biomaterials, biotechnol., and biomedicine.
    9. 9
      Schaffner, M., Ruhs, P. A., Coulter, F., Kilcher, S., and Studart, A. R. (2017) 3D printing of bacteria into functional complex materials. Sci. Adv. 3, eaao6804  DOI: 10.1126/sciadv.aao6804
      There is no corresponding record for this reference.
    10. 10
      Gonzalez, L. M., and Voigt, C. A. (2019) Resilient living materials built by printing bacterial spores. bioRxiv 537571.
      There is no corresponding record for this reference.
    11. 11
      Nguyen, P. Q. (2017) Synthetic biology engineering of biofilms as nanomaterials factories. Biochem. Soc. Trans. 45, 585– 597,  DOI: 10.1042/BST20160348
      11
      Synthetic biology engineering of biofilms as nanomaterials factories
      Nguyen, Peter Q.
      Biochemical Society Transactions (2017), 45 (3), 585-597CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
      Bottom-up fabrication of nanoscale materials has been a significant focus in materials science for expanding our technol. frontiers. This assembly concept, however, is old news to biol. - all living organisms fabricate themselves using bottom-up principles through a vast self-organizing system of incredibly complex biomols., a marvelous dynamic that we are still attempting to unravel. Can we use what we have gleaned from biol. thus far to illuminate alternative strategies for designer nanomaterial manufg. In the present review article, new synthetic biol. efforts toward using bacterial biofilms as platforms for the synthesis and secretion of programmable nanomaterials are described. Particular focus is given to self-assembling functional amyloids found in bacterial biofilms as re-engineerable modular nanomol. components. Potential applications and existing challenges for this technol. are also explored. This novel approach for repurposing biofilm systems will enable future technologies for using engineered living systems to grow artificial nanomaterials.
    12. 12
      Chen, A. Y., Zhong, C., and Lu, T. K. (2015) Engineering living functional materials. ACS Synth. Biol. 4, 8– 11,  DOI: 10.1021/sb500113b
      12
      Engineering Living Functional Materials
      Chen, Allen Y.; Zhong, Chao; Lu, Timothy K.
      ACS Synthetic Biology (2015), 4 (1), 8-11CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      A review. Natural materials, such as bone, integrate living cells composed of org. mols. together with inorg. components. This enables combinations of functionalities, such as mech. strength and the ability to regenerate and remodel, which are not present in existing synthetic materials. Taking a cue from nature, we propose that engineered 'living functional materials' and 'living materials synthesis platforms' that incorporate both living systems and inorg. components could transform the performance and the manufg. of materials. As a proof-of-concept, we recently demonstrated that synthetic gene circuits in Escherichia coli enabled biofilms to be both a functional material in its own right and a materials-synthesis platform. To demonstrate the former, we engineered E. coli biofilms into a chem.-inducer-responsive elec. switch. To demonstrate the latter, we engineered E. coli biofilms to dynamically organize biotic-abiotic materials across multiple length scales, template gold nanorods, gold nanowires, and metal/semiconductor heterostructures, and synthesize semiconductor nanoparticles. Thus, tools from synthetic biol., such as those for artificial gene regulation, can be used to engineer the spatiotemporal characteristics of living systems and to interface living systems with inorg. materials. Such hybrids can possess novel properties enabled by living cells while retaining desirable functionalities of inorg. systems. These systems, as living functional materials and as living materials foundries, would provide a radically different paradigm of materials performance and synthesis-materials possessing multifunctional, self-healing, adaptable, and evolvable properties that are created and organized in a distributed, bottom-up, autonomously assembled, and environmentally sustainable manner.
    13. 13
      Tamsir, A., Tabor, J. J., and Voigt, C. A. (2011) Robust multicellular computing using genetically encoded NOR gates and chemical ’wires. Nature 469, 212– 215,  DOI: 10.1038/nature09565
      13
      Robust multicellular computing using genetically encoded NOR gates and chemical "wires'
      Tamsir, Alvin; Tabor, Jeffrey J.; Voigt, Christopher A.
      Nature (London, United Kingdom) (2011), 469 (7329), 212-215CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Computation underlies the organization of cells into higher-order structures, for example during development or the spatial assocn. of bacteria in a biofilm. Each cell performs a simple computational operation, but when combined with cell-cell communication, intricate patterns emerge. Here the authors study this process by combining a simple genetic circuit with quorum sensing to produce more complex computations in space. They construct a simple NOR logic gate in Escherichia coli by arranging two tandem promoters that function as inputs to drive the transcription of a repressor. The repressor inactivates a promoter that serves as the output. Individual colonies of E. coli carry the same NOR gate, but the inputs and outputs are wired to different orthogonal quorum-sensing 'sender' and 'receiver' devices. The quorum mols. form the wires between gates. By arranging the colonies in different spatial configurations, all possible two-input gates are produced, including the difficult XOR and EQUALS functions. The response is strong and robust, with 5- to >300-fold changes between the 'on' and 'off' states. This work helps elucidate the design rules by which simple logic can be harnessed to produce diverse and complex calcns. by rewiring communication between cells.
    14. 14
      Osmekhina, E., Jonkergouw, C., Schmidt, G., Jahangiri, F., Jokinen, V., Franssila, S., and Linder, M. B. (2018) Controlled communication between physically separated bacterial populations in a microfluidic device. Commun. Biol. 1, 97,  DOI: 10.1038/s42003-018-0102-y
      14
      Controlled communication between physically separated bacterial populations in a microfluidic device
      Osmekhina Ekaterina; Jonkergouw Christopher; Schmidt Georg; Linder Markus B; Jahangiri Farzin; Jokinen Ville; Franssila Sami
      Communications biology (2018), 1 (), 97 ISSN:.
      The engineering of microbial systems increasingly strives to achieve a co-existence and co-functioning of different populations. By creating interactions, one can utilize combinations of cells where each population has a specialized function, such as regulation or sharing of metabolic burden. Here we describe a microfluidic system that enables long-term and independent growth of fixed and distinctly separate microbial populations, while allowing communication through a thin nano-cellulose filter. Using quorum-sensing signaling, we can couple the populations and show that this leads to a rapid and stable connection over long periods of time. We continue to show that this control over communication can be utilized to drive nonlinear responses. The coupling of separate populations, standardized interaction, and context-independent function lay the foundation for the construction of increasingly complex community-wide dynamic genetic regulatory mechanisms.
    15. 15
      Tay, P. K. R., Basavanna, A. M., and Joshi, N. S. (2018) Repurposing bacterial extracellular matrix for selective and differential abstraction of rare earth elements. Green Chem. 20, 3512,  DOI: 10.1039/C8GC01355A
      15
      Re-purposing bacterial extracellular matrix for selective and differential abstraction of rare earth elements
      Tay, Pei Kun R.; Manjula-Basavanna, Avinash; Joshi, Neel S.
      Green Chemistry (2018), 20 (15), 3512-3520CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
      The rare earth elements (REEs) play crit. roles in modern consumer electronics and clean technologies, but unpredictable supply and environmentally unsustainable extn. practices have spurred efforts to develop green methods of recovering the metals from waste streams. In this regard, the authors have re-purposed a bacterial extracellular matrix (ECM) for selective and differential abstraction of REEs. Herein, the curli amyloid fibers in E. coli biofilms are genetically modified to display lanthanide binding tags (LBTs). The curli-LBT filters showed lanthanide specificity in the presence of other metals, with a preference for binding several high-value heavy REEs. Bound lanthanides were readily recovered using a dil. acid wash, and the filters could be re-used for multiple cycles of sorption and desorption with minimal loss of efficiency. The authors' engineered biofilm-derived filters provide a rapid, selective and scalable method for REE sepn. that is more robust compared to conventional cell-based sorbents, and this platform could be adapted to recover other precious metals or commodities.