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GeneGuard: A Modular Plasmid System Designed for Biosafety

Oliver Wright^†^‡
,
Mihails Delmans^†^‡
,
Guy-Bart Stan^†^‡
, and
Tom Ellis^*^†^‡

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† Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, United Kingdom

‡ Department of Bioengineering, Imperial College London, London SW7 2AZ, United Kingdom

*Phone: +44 0 20 7594 7615. Fax: +44 0 20 7594 9817. Email: [email protected]

Cite this: ACS Synth. Biol. 2015, 4, 3, 307–316

Publication Date (Web):May 13, 2014

https://doi.org/10.1021/sb500234s

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Abstract

Synthetic biology applications in biosensing, bioremediation, and biomining envision the use of engineered microbes beyond a contained laboratory. Deployment of such microbes in the environment raises concerns of unchecked cellular proliferation or unwanted spread of synthetic genes. While antibiotic-resistant plasmids are the most utilized vectors for introducing synthetic genes into bacteria, they are also inherently insecure, acting naturally to propagate DNA from one cell to another. To introduce security into bacterial synthetic biology, we here took on the task of completely reformatting plasmids to be dependent on their intended host strain and inherently disadvantageous for others. Using conditional origins of replication, rich-media compatible auxotrophies, and toxin–antitoxin pairs we constructed a mutually dependent host-plasmid platform, called GeneGuard. In this, replication initiators for the R6K or ColE2-P9 origins are provided in trans by a specified host, whose essential thyA or dapA gene is translocated from a genomic to a plasmid location. This reciprocal arrangement is stable for at least 100 generations without antibiotic selection and is compatible for use in LB medium and soil. Toxin genes ζ or Kid are also employed in an auxiliary manner to make the vector disadvantageous for strains not expressing their antitoxins. These devices, in isolation and in concert, severely reduce unintentional plasmid propagation in E. coli and B. subtilis and do not disrupt the intended E. coli host’s growth dynamics. Our GeneGuard system comprises several versions of modular cargo-ready vectors, along with their requisite genomic integration cassettes, and is demonstrated here as an efficient vector for heavy-metal biosensors.

KEYWORDS:

Current microbial synthetic biology systems are predominantly built for use in contained bioreactors, for example, for the production of valuable compounds. (1) Other applications, by their very nature, are proposed for use outside the confines of a laboratory (e.g., biosensors, (2) bioremediation, (3) and biomining). (4) This leads to understandable concern over the release of genetically modified microbes (GMMs), including their unchecked proliferation and the possibility of “genetic pollution”, (i.e., the undesired establishment of synthetic genetic material in other organisms).

In the past three decades, researchers have developed a catalogue of approaches for engineering microbes with the intention to address such GMM biosafety issues (e.g., “kill-switches”, auxotrophies). Two recent reviews described the strengths and weaknesses of these methods with respect to synthetic biology (5, 6) and highlighted how complete genomic recoding (7) and “xenobiology” (8) may offer solutions for the future. However, given that such technologies are in their infancy, a here-and-now solution is essential for biosensors and related GMM projects that are currently underway. In this work, we seek to fulfill this need by combining existing biosafety devices into what we believe is a robust strategy to counter unwanted horizontal gene transfer (HGT) of synthetic DNA.

Working with E. coli, we describe a plasmid-based system linked to its intended host strain via three separate mechanisms to ensure system redundancy. Although plasmids are extensively used in both synthetic biology and industrial biotechnology, as mobile genetic elements they are inherently an environmental biosafety concern (e.g., antibiotic resistance spread). Despite this, they offer two crucial advantages over the alternative strategy of placing synthetic genes into the host microbe genome: (i) they are easier to construct, test and tune compared to genomically integrated DNA; and (ii) via imperfect retention and/or a lack of selection, (9) they have a limited half-life when deployed into an environment. Furthermore, synthetic DNA placed within plasmids is not flanked by native genomic sequences, as it is with genomically integrated constructs. Such native flanking sequence gives the potential for synthetic DNA to homologously recombine into unmodified strains that possess significant genome homology to the intended host.

Our plasmid system, called GeneGuard, focuses on three device classes that lead to host-plasmid mutual dependency: (i) a conditional origin of replication (COR), in which the requisite plasmid replication initiator protein is provided in trans; (ii) complementation of an introduced host auxotrophy, with compatibility for use in common rich-media; and (iii) plasmid-encoding of a broad-spectrum toxin to select against plasmid spread by making the plasmid DNA itself disadvantageous to maintain by a wild-type bacterium. Importantly, GeneGuard does not need to utilize antibiotic resistance cassettes.

We assemble combinations of these devices using the Standard European Vector Architecture (SEVA (10)), and tune their expression using BIOFAB bicistronic domains. (11) The result is a collection of new “secure” plasmids that are able to replace those used as the vector DNA for most E. coli synthetic biology projects. We examine here their efficiency for use in the lab and provide a preliminary assessment of their compatibility for use in soil. Selected devices are also examined for their effects on host growth rate, and their ability to inhibit horizontal gene transfer is assessed via electroporation into E. coli and natural transformation into supercompetent Bacillus subtilis. (12) Finally, as a proof-of-principle, we show that heavy-metal biosensors built and hosted on commonly used plasmid vectors perform as well, if not better, in the GeneGuard system.

Results and Discussion

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To build the GeneGuard system we took three biosecurity device classes (CORs, auxotrophy complementation and toxin–antitoxin pairs), tested their functionality in E. coli, and combined them into a set of customizable plasmids built upon the modular SEVA plasmid architecture. (10)

Conditional Origins of Replication (CORs)

Using pSEVA111 (10) as a starting point, constitutive mRFP1 reporter vectors containing either the R6K (13) or ColE2 (-P9) (14) COR were built (pSEVA117Rb and pSEVA177Rb) and transformed into three different tunable plasmid copy number “DIAL” strain variants. (15) As a proxy for confirming copy number control, fluorescent output was assessed by flow cytometry (Figure 1; see Supporting Information Table 1 for comprehensive plasmid and strain details). Low-level expression of the replication initiator proteins for R6K and ColE2 (π and RepA, respectively) in DIAL strain AB resulted in near background levels of observable fluorescence. Medium-level expression (DIAL strain EI) gave an output higher than that observed from a low-copy number RK2 origin control (pSEVA127Rb; ∼4–15 per cell (16)), while R6K/ColE2 plasmids hosted under a high level of π/RepA expression (DIAL strain JK; previously unpublished) resulted in equivalent or greater fluorescence than that observed from a high-copy number pUC origin control (pSEVA167Rb; ∼100 per cell (17, 18)). These results are consistent with the original DIAL strain data, which correlated well to quantitative PCR estimation of plasmid copy number. (15) Furthermore, the inability of these CORs to propagate in the absence of π or RepA was confirmed through repeatedly unsuccessful attempts to transform wild-type E. coli (i.e., no colonies were obtained unless DIAL strains were used). This confirms that COR plasmid replication is dependent on a specific host, thereby inhibiting establishment in other microbes.

Figure 1. Flow cytometry of DIAL strains hosting COR reporter plasmids. (a) Schematic of COR plasmid dependence on host DIAL strain, where plasmid copy number is tuned by the ribosome binding site (RBS) strength of the replication initiator protein transcript. (b) DIAL strains constitutively expressing both π and RepA at low (AB), medium (EI) and high (JK) levels were transformed with mRFP1 reporter plasmids containing the R6K (pSEVA117Rb) or ColE2 (pSEVA177Rb) COR, and fluorescence assessed at mid log growth phase by flow cytometry. Low-copy RK2 (pSEVA127Rb) and high-copy pUC (pSEVA167Rb) origins were also profiled as controls. Median fluorescence values and robust coefficient of variation (rCV) are indicated beneath each plot (n = 4 biological repeats; representatives shown). W/T, wild-type *E. coli* MC1061 used for DIAL strain construction; au, arbitrary units; X-axis, side scatter; RBS EI, RBS E for π, RBS I for RepA (see Supporting Information Table 1 for more detail).

Rich-media Compatible Auxotrophs

The use of antibiotic resistance genes is inappropriate for environmental applications not only due to concerns over HGT but also because of the impracticality of continually applying a selective agent to a GMM-deployed area. An alternative strategy for host-plasmid dependency is auxotrophic complementation. Common auxotrophies (e.g., amino acid biosynthesis knockouts) usually require defined minimal-media to ensure key metabolite absence, and typically result in a decreased growth rate. Of greater utility are auxotrophs compatible with rich-media (e.g., LB), such as knockouts in E. coli for thymidine (ΔthyA) (19) or diaminopimelic acid (DAP; ΔdapA). (20) Rich-media supplementation with small quantities of these metabolites supports the growth of knockouts, as does complementation via introduction of a plasmid carrying the relevant knocked-out gene.

Using the DIAL strains as a starting chassis, ΔthyA or ΔdapA auxotrophs were created using the λ Red recombinase method, (21, 22) and integrants were verified by colony PCR. These auxotrophs were unable to grow in LB medium unless supplemented with the appropriate key metabolite. To complement these knockouts, the thyA and dapA open reading frames were amplified from E. coli MC1061 and separately inserted into the R6K and ColE2 COR reporter plasmids (after SEVA (10) and BioBrick (23) incompatible restriction sites had been removed, giving pSEVA117RbT or D, pSEVA177RbT or D). BIOFAB constitutive promoters and bicistronic designs (BCDs), configured for reliable levels of gene expression, (11) were used to heuristically tune thyA and dapA expression. Promoter P14 coupled to BCD7 was picked as a prospective medium-strength combination and was found to be suitable for low-copy DIAL strain auxotroph complementation. In medium or high-copy DIAL strain auxotrophic variants; however, transformants were not obtainable. A second iteration using the weaker promoter P12 (∼65% strength of P14) was found to exhibit wild-type growth characteristics in all DIAL strains used, indicating that a reasonable range of thymidylate synthase (thyA) or 4-hydroxy-tetrahydrodipicolinate synthase (dapA) is sufficient for key metabolite production.

The stability of our thyA/dapA plasmids was assessed at a low-copy number (DIAL strain AB), with their complementation of the introduced auxotrophy acting as sole selection pressure (Figure 2b). In the absence of selection, the probability of each daughter cell receiving a plasmid from its mother is not sufficient to support indefinite propagation in a population over time. This was confirmed by the R6K and ColE2 COR reporter plasmids (pSEVA117Rb and pSEVA177Rb) showing near complete depletion after ∼100 cell divisions. In contrast, when auxotrophy complementation was employed, all cells analyzed retained the reporter plasmid (pSEVA117RbT or D, pSEVA177RbT or D). The previously used low-copy number RK2 origin control (pSEVA127Rb) was retained by approximately half of the population under the same conditions, while the high-copy pUC origin reporter plasmid (pSEVA167Rb) was lost from the population after ∼60 to 70 divisions, its presence likely placing an unfavorable burden on the host due to the energetic cost of its high replication rate.

While these two auxotrophy systems provide an antibiotic-free method of selecting for plasmids, their stability can easily be disturbed by the addition to the media of the relevant key metabolite (Figure 2c; thyA⁺ plasmids are lost more quickly than dapA⁺). This indicates the importance of choosing auxotrophies based on metabolites that are absent from the intended application environment. As an example, the performance of the ΔthyA and ΔdapA E. coli DIAL strains in garden soil was investigated. Soil was taken and used as a supplement for overnight cultures of auxotrophic medium-copy DIAL strains (EI) in SOB (Figure 2d). Both ΔthyA and ΔdapA strains grew poorly in nonsterile soil, with ΔdapA resulting in the fewest E. coli colonies. (Observation of larger, noncoliform colonies indicates the presence of additional kanamycin-resistant microbes in the soil used.) When supplemented with either the appropriate key metabolite or complementation plasmid (e.g., pSEVA177RbT or D), knockout strain growth density returned to wild-type levels. When sterile soil was used (Supporting Information Figure 1) in order to remove any potential competition effect that native microbes may have on key metabolite utilization, the ΔdapA strain still failed to thrive, implying that there is insufficient environmental DAP to support knockout growth. (24, 25) The growth of ΔthyA, however, was restored, indicating the presence of adequate thymidine nucleoside in the sterilized soil sample. Although this indicates potential for the eventual disruption of plasmid propagation, such imperfect retention may actually be preferred from a biosafety standpoint. Gradual plasmid loss, when combined with a host fitness deficit, should lead to GMMs that survive for months in the environment, rather than years. This is preferable for reducing the long-term chance of genetic pollution, and could theoretically be tuned for specific applications. For example, an auxotrophy based on an abundant environmental metabolite could be used with contained biosensors (e.g., those housed in a pregnancy-test-like device (2)), thereby ensuring a rapid loss of plasmid retention pressure in the event of GMM leakage.

Figure 2. Plasmid stability in auxotrophic strains. (a) Schematic of DIAL strain auxotroph (Δ*thyA* or Δ*dapA*) dependence on complementing plasmid (*thyA*⁺ or *dapA*⁺). Orange arrow indicates complementation of knocked-out gene (remnants represented by orange bars on chromosome). (b) Stability assay measuring the proportion of colonies that retain their plasmid in low-copy DIAL strain AB after ∼100 generations in liquid LB without antibiotic selection (n = 4 biological repeats; error bars = standard deviation). (c) Assay as above, but plated on agar containing key metabolite to assess plasmid stability when auxotrophic pressure is removed (n = 4 biological repeats; error bars = standard deviation). (d) Assessment nonsterile soil’s ability to provide key metabolite to auxotrophs when added to liquid SOB and incubated overnight (dilution series subsequently plated on kanamycin-containing LB agar to suppress growth of other soil microbes). kanR, kanamycin resistance cassette; dark purple bars, FRT (flippase recognition target); thyd., thymidine; DAP, diaminopimelic acid; KO, knockout; R6K (pSEVA117Rb), ColE2 (pSEVA177Rb), R6K-thyA (pSEVA117RbT), ColE2-thyA (pSEVA177RbT), R6K-dapA (pSEVA117RbD), ColE2-dapA (pSEVA177RbD), low RK2 (pSEVA127Rb), high pUC (pSEVA167Rb) (see Supporting Information Table 1 for more detail).

Toxin–Antitoxin Systems As a Negative Selection Pressure

A GMM that undergoes environmental deployment may contain synthetic genes that are advantageous for other organisms to acquire. To further reduce the likelihood of plasmid acquisition beyond a specified host, DNA-encoded broad-spectrum toxins were investigated as a means of exerting negative selection pressure on wild-type microbes. With plasmid-encoded toxins, host immunity is provided in trans via genomic integration of the cognate antitoxin. Full dependency on the expression or function of a toxin–antitoxin pair is a major flaw in previous “kill-switch” designs, as an inability to protect against inactivating mutations ultimately leads to safety system failure. (5, 26) In GeneGuard, a toxin–antitoxin pair is instead employed in an auxiliary manner, that is, continued function is not critical to the overall integrity of the system.

As toxin activity is required to be broad-spectrum to work against a variety of environmental microbes, a broad-host-range constitutive promoter was used to maximize the likelihood of expression, and the toxins ζ (from Streptococcus pyogenes plasmid pSM19035) (27, 28) and Kid (from E. coli plasmid R1) (29) were investigated. These proteins are reported to be growth-inhibitory (i.e., bacteriostatic) when expressed in Gram-positive, Gram-negative, and even some eukaryotic hosts. It is important to note that the term “toxin” relates to their activity as cytosolic enzymes: ζ interferes with the beginning of peptidoglycan synthesis, (30) while Kid is a sequence-specific endoribonuclease. (31) The poisoning of nearby organisms is therefore highly unlikely, as any released enzyme would require cellular internalization before toxicity could occur; its degradation is more probable.

His-tagged antitoxin genes for ε (27) or Kis (29) were paired with promoter P1 and ThrA-BCD2 (11) and genome-integrated as per previously (i.e., auxotrophs were also simultaneously created; Figure 3a). While integration fixes antitoxin gene copy number, plasmid-encoded toxin levels will vary depending on the host DIAL strain used. A balance is therefore required so that toxin gene dosage at a high plasmid copy number does not overwhelm the available antitoxin, while maintaining an adequate expression of toxin such that a low-copy dosage remains toxic to wild-type cells lacking the antidote. This was achieved through tuning toxin expression levels with a designed, E. coli and B. subtilis compatible, spoVG RBS (32) paired with a similarly compatible constitutive Pveg2 promoter. His-tagged ζ and Kid genes were inserted into the thyA/dapA complementation plasmids (containing the ColE2 COR) so that both the auxotrophy complementation and toxin open reading frames converged upon a bidirectional terminator flanked by synthetic insulating spacers. (33)

The use of spoVG2 gave tolerable levels of ζ in all antitoxin-expressing DIAL strains. Kid expression, however, required down-tuning for its plasmid to be acceptable to the high-copy DIAL strain JK (the spoVG5 promoter, at ∼27% of the strength (32) of spoVG2, was settled upon). Both the ζ and Kid plasmids (pSEVA177RbTZh or TKh, DZh, or DKh), when hosted by their respective antitoxin/auxotrophic DIAL strains (AB, EI, or JK), did not perturb the growth rate from that of the wild-type (Figure 3b; Supporting Information Figure 2). Expression of antitoxin was confirmed via Western blot (Figure 3c) with toxin expression only just visible for both types in the high-copy DIAL strain JK. The strong antitoxin bands observed indicate that greater expression of both toxins should be possible: ζ is neutralized by ε at a 1:1 ratio, (30) while two molecules of Kid are neutralized by each Kis. (34) Greater expression was not found to be possible for Kid, however, as demonstrated by the need to tune its translation down with spoVG5.

To confirm toxin activity in cells lacking antitoxin, the above plasmids were transformed into the low, medium and high-copy number auxotrophic DIAL strains that lacked the relevant antitoxin gene, and as intended, no colonies were immediately obtainable. After prolonged incubation for ∼72 h, however, thousands of small colonies appeared on low-copy DIAL strain AB plates hosting the Kid toxin. This possibly indicates insufficient Kid expression for complete bacteriostasis. In addition, when the selection pressure of auxotrophy complementation was removed through the use of prototrophic DIAL strains, several healthy colonies were obtained from each toxin plasmid transformation. This was due to deleterious recA-mediated homologous recombination between two similarly orientated pVeg promoters within our test plasmids, leading to toxin gene removal (pVeg also drove mRFP1 expression). This event was not seen in our auxotrophic DIAL strains, as such recombination also leads to the deletion of the essential complementation gene (see Figure 3a).

To assess toxin activity in a Gram-positive bacterium, toxin genes were transferred to a SEVA shuttle backbone containing a dual replication origin (pUC and pTHT15) and a chloramphenicol resistance cassette compatible for use in both E. coli and B. subtilis. (35) These constructs (pSEVA3b6Zh or Kh) contained no mRFP1 reporter, eliminating the previous source of recombination-mediated toxin deletion. To simulate a transformation that is more environmentally realistic than electroporation, a competence-inducible B. subtilis strain (SCK6 (12)) was used. While electroporation of E. coli DH10B gave no colonies for either the ζ or Kid toxin plasmids (Figure 3d), several ζ colonies and hundreds of Kid colonies resulted when transforming B. subtilis SCK6. The lack of Kid toxicity in B. subtilis SCK6 is likely due to the pre-existing genomic presence of a Kis antitoxin homologue, such as YdcD. (36) Taken together, the data presented reinforces the futility of relying on toxin integrity over time, and supports our approach of only utilizing kill-switch devices in an auxiliary role. Table 1 summarizes the ability of the described devices to influence plasmid propagation.

Figure 3. Use of ζ–ε and Kid-Kis toxin–antitoxin pairs. (a) Schematic of how a toxin-encoding plasmid may prove deleterious if taken up by wild-type cells, while the specified host cell possesses genome-encoded immunity. (b) Growth curves of high-copy DIAL strain JK with various combinations of Δ*dapA* auxotrophy, chromosomally integrated ε or Kis antitoxin, and plasmid-encoded ζ or Kid toxin (ColE2 COR used; n = 3 biological repeats; error bars = standard deviation). For Δ*thyA* auxotrophy, and other plasmid copy numbers, see Supporting Information Figure 2. (c) Western blot of various DIAL strains constitutively expressing integrated ε (11.5 kDa) or Kis (10.2 kDa) antitoxins alone, as well as with plasmid-encoded ζ (33.2 kDa) or Kid (12.7 kDa) toxins. All toxins/antitoxins are His-tagged at the C-terminus; putative toxin bands are arrowed. (d) Transformation assessment of ability of wild-type cells to maintain toxin plasmid in the absence of integrated antitoxin. W/T, wild-type; antiT, antitoxin; WM, weight marker; dapA (pSEVA177RbD); dapA-ζ (pSEVA177RbDZh); dapA-Kid (pSEVA177RbDKh); control plasmid (pSEVA3b61); ζ plasmid (pSEVA3b6Zh); Kid plasmid (pSEVA3b6Kh).

Table 1. Summary of COR, Auxotrophy Complementation and Toxin Device Effects on Host Cell Propagationa

	pSEVA3b61 Control	pSEVA117Rb R6K COR	pSEVA177Rb ColE2 COR	pSEVA3b6Zh ζ toxin	pSEVA3b6Kh Kid toxin	pSEVA177RbT ColE2 + thyA	pSEVA177RbD ColE2 + dapA	pSEVA177RbDZh ColE2 + dapA + ζ	pSEVA177RbDKh ColE2 + dapA + Kid
E. coli MC1061	>1000 (n = 3)	0 (n = 2)	0 (n = 2)	<10 (n = 2)	<10 (n = 2)	−	−	−	−
E. coli DIALb	>1000 (n = 2)	>1000 (n = 3)	>1000 (n = 3)	<10 (n = 4)	<10 (n = 2)	>1000 (n = 2)	>1000 (n = 2)	<10 (n = 3)c	<100 (n = 3)c
E. coli DIAL auxob	−	−	−	−	−	>1000 (n = 5)	>1000 (n = 5)	0 (n = 6)	0 (n = 6)
E. coli DIAL auxo/antitoxinb	−	−	−	>1000 (n = 2)	>1000 (n = 2)	>1000 (n = 2)	>1000 (n = 1)	>1000 (n = 6)	>1000 (n = 6)
B. subtilis SCK6	>100 (n = 3)	0 (n = 2)d	<10 (n = 2)d	<10 (n = 3)	>100 (n = 3)	−	−	−	−

Approximate number of colonies resulting from transformation of plasmid containing the various devices into each cell line.

Low, medium or high plasmid copy number DIAL strains gave similar results for each plasmid (strains with cognate auxotrophies/antitoxins used where appropriate).

Homologous recombination led to deletion of toxin gene.

pSEVA3b17Rb or pSEVA3b77Rb used respectively for testing R6K or ColE2 in B. subtilis due to antibiotic resistance cassette compatibility. Dashes indicate experiment not performed as considered unnecessary. For full plasmid and strain details, see Supporting Information Table 1.

GeneGuard Genomic and Vector Cassettes

Following assessment of the individual devices, various COR, auxotrophy, and toxin–antitoxin combinations were arranged into a complete GeneGuard system (Figure 4). A genomic integration cassette was designed to serve as a PCR template for the λ Red-mediated genomic insertion method, and consists of genes for the COR replication initiator, an antitoxin and a kanamycin selection cassette, all flanked by the necessary 5′ and 3′ homology arms for thyA or dapA knockout creation. A low-copy RK2 origin is used in this plasmid to minimize gene dosage problems (excess antitoxin production was itself found to exhibit toxicity), and a constitutive mRFP1 marker allows for easy identification of false-positives during the integration procedure (i.e., template plasmid carryover). Once a knockout is verified, P1 transduction can be performed if desired, and the FRT-flanked kanamycin selection cassette may be excised using the pCP20-encoded FLP recombinase. (21) Note that the kanamycin resistance gene is not strictly necessary when integrating at thyA, as trimethoprim may instead be used as a counter-selection agent. (19)

The accompanying vector cassette consists of a COR region and toxin and auxotrophy complementation genes. With a total size of ∼2.7 kbp, this compact cassette may be directly amplified by PCR and used to simultaneously replace the replication origin and resistance gene of a pre-existing plasmid (i.e., retrofitting) or the vector cassette itself can be used as a readymade plasmid backbone for the insertion of cargo DNA. To ease initial plasmid manipulation in standard cloning strains, a removable antibiotic resistance cassette is also included in the multicloning site of the vector cassette backbone.

Figure 4. Schematic of the GeneGuard system. The genomic cassette (a) consists of replication initiator, antitoxin and FRT-bound kanamycin resistance genes, flanked by ∼280 to 500 bp of 5′/3′ UTR sequence from the *thyA* or *dapA* genes (total cassette size of ∼3.6–3.8 kbp). The vector cassette (b) hosts cargo DNA via a pUC18-derived multicloning site that contains a removable antibiotic resistance gene between the PacI and AvrII sites. To retrofit existing plasmids, the COR/toxin/auxotrophy cassette (c) may be PCR-amplified and swapped with the existing origin/antibiotic resistance region. After construction, GeneGuard-derived plasmids are dependent on host cells that contain the requisite genomic cassette (d).

Using the GeneGuard System for Heavy-Metal Biosensors

The GeneGuard system must satisfy two criteria for use in environmental synthetic biology applications: (i) it must increase biosafety but (ii) not disrupt application functionality. To demonstrate that our plasmids fit the second criterion, we took previously described arsenic, mercury, and copper biosensors (37) and compared their performance when hosted on their original backbone to that of two different medium-copy GeneGuard plasmids (Figure 5). When the pD1K backbone (dapA⁺, R6K COR, Kid toxin) is used, fluorescence output at all heavy-metal concentrations is beneficially elevated in comparison to the original medium-copy number pSB3K3 BioBrick plasmid. When pT7Z (thyA⁺, ColE2 COR, ζ toxin) is used, the biosensors have a dose response profile more equivalent to that of the original pSB3K3 backbone. This proves that the GeneGuard system does not hamper cargo function, and hence that it could be used for real-world deployment.

The useful but unexpected increase in dynamic signal range for pD1K-hosted biosensors (see Supporting Information Figure 3) is likely due to greater than expected π replication initiator production, resulting in a higher than desired plasmid copy number. This illustrates the importance of the genomic cassette’s architecture: whereas in the original DIAL strains the replication initiator genes were placed in disparate genomic regions, here they were inserted immediately downstream of the native thyA or dapA promoter region and may therefore suffer from transcriptional read-through. Inversion of the replication initiator gene in a future iteration of the GeneGuard genomic cassette would address this.

Our GeneGuard system, while simple in concept, is sturdier in design when compared to other complicated biosecurity solutions. (38, 39) Through the use of three distinct mechanisms, it has redundancy in its design and is highly unlikely to provide any benefit to wild-type cells, therefore limiting the potential for genetic pollution. A COR sequence on its own is of little use, unless receiving microbes already host similar initiator-encoding plasmids (and even then plasmid incompatibility will likely result). The acquisition of a constitutive auxotrophy complementation gene also provides little utility; it may lead to greater flux through a pathway, but as thymidine and DAP biosynthesis genes are already pervasive, this is unlikely to confer a significant benefit. The toxins have been selected specifically to produce a negative selection pressure, and even if their activity is neutralized via mutation or through the presence of a pre-existing antitoxin, no evolutionary advantage will ensue. While this version of GeneGuard is designed to work in E. coli, application to other environmentally relevant bacteria is possible. For example, thymidine and DAP auxotrophies have been experimented with in Pseudomonas fluorescens (24, 25) and P. putida (40) for bioremediation roles, while B. subtilis required the deletion of two discontiguous thymidylate synthetase genes to create a thymidine auxotroph. (41) It is important to note, however, that each species requires context-specific optimization of GeneGuard device expression and function, a nontrivial task if plasmid construction and propagation during this optimization relies on using E. coli cloning strains.

GeneGuard, as presented here, represents the state-of-the-art for E. coli plasmid biosecurity. In our opinion, the best combination of parts profiled would be the ColE2 COR combined with ΔdapA complementation (soil lacks sufficient DAP) and the ζ toxin (effective against E. coli and B. subtilis). Our system could further be improved to limit successful HGT by refactoring vector parts to have minimal homology to all known microbial genomes (e.g., codon shuffling of the auxotrophy complementation genes) or plasmids, and through the future addition of alternative parts into the modular set. In addition, integration of the replication initiator and antitoxin genes at different genomic loci would further decrease the chance of both parts transposing to, or with, the synthetic plasmid into other cells. This, however, would require additional genomic manipulation. In conclusion, adoption of GeneGuard for environmental synthetic biology would be a beneficial move, as common biosafety concerns can be addressed without detriment to end-use applications. Whether this satisfies local and/or supranational regulations, however, is an ongoing debate. A recent workshop (42) held on this subject, covering past examples and current regulatory issues, discusses and summarizes the prospects and hurdles faced for future deployment requests of GMMs.

Figure 5. GeneGuard system applied to heavy-metal biosensors. (a) Schematic of biosensors inserted into GeneGuard plasmids. Arsenic relieves ArsR repression of ParsR; mercury relieves MerR repression of PmerT; and copper enables CusR activation of PcusC. Each of these promoters is linked to the reporter GFPmut3b. (b) Dose response curves in *E. coli* DH10B for the arsenic biosensor in its original plasmid (pSB3K3 contains a medium-copy p15A origin, requires kanamycin selection), and its performance when ported to GeneGuard variants pD1K (*dapA*, R6K COR, Kid toxin; no antibiotic selection used) and pT7Z (*thyA*, ColE2 COR, ζ toxin; no antibiotic selection used) with the requisite genomic cassettes inserted to support a medium-copy plasmid number (n = 4 biological repeats; error bars = standard deviation). (c) Dose response curves for the mercury biosensor, as per part b. (d) Dose response curves for the copper biosensor, as per part b. W/T, wild-type; au, arbitrary units; WHO, World Health Organization. (43) For low and high-copy GeneGuard plasmid results, see Supporting Information Figure 3.

Methods

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Media and General Materials

Microbes were propagated in LB broth/agar, supplemented as appropriate with thymidine (20 mg/mL stock prepared in water) or diaminopimelic acid (DAP; 20 mg/mL stock prepared in water, with 10 M NaOH added dropwise until solute dissolved) to a final concentration of 50 μg/mL. (44-46) (Thymine, despite a previous report, (19) was unable to support ΔthyA cells.) Ampicillin was used at 100 μg/mL; kanamycin at 25 μg/mL; and chloramphenicol at 6 μg/mL. Sterile polystyrene 96-well plates from Corning B.V. Life Sciences were used for all assays, and sealed with Breathe-Easy sealing membrane (Sigma-Aldrich Company Ltd.) prior to any incubation step. B. subtilis SCK6 (12) was purchased from the Bacillus Genetic Stock Center (http://www.bgsc.org/).

Device Construction

Oligos were ordered from Integrated DNA Technologies BVBA, and larger DNA pieces as GeneArt Strings from Life Technologies Ltd. Phusion High-Fidelity DNA polymerase (20 μL reactions; New England Biolabs (U.K.) Ltd.) was used according to the manufacturer’s instructions for part construction, along with touchdown PCR (47) and overlap extension PCR (48) for device assembly/point mutations (for full sequence details and GenBank accession numbers, see Supporting Information Table 1). For colony PCR, REDTaq ReadyMix (10 μL reactions; Sigma-Aldrich Company Ltd.) was used according to the manufacturer’s instructions on colony scrapings from sterile toothpicks. Sequence verification was performed by Source BioScience Plc.

Flow Cytometry

Using 96-well plates, 2 μL of overnight culture was used to seed 200 μL of fresh LB (with ampicillin) and incubated to mid log phase (30 °C, ∼710 rpm, for ∼3 h). After dilution in water, samples were analyzed using a FACScan (Becton Dickinson Co.) that had been upgraded by Cytek Development Inc. and coupled to an Automated Machine Sampler system (Cytek Development Inc.). Excitation of mRFP1 was at 561 nm, with emission monitored at 615/25 nm. Data was collected using CellQuest Pro (v5.1.1; Becton Dickinson Co.), and processed using FlowJo (v7.6.5; Tree Star Inc.).

Genomic Integration

Genomic integration was performed as previously described. (21) For each reaction, 166 μL overnight culture of desired host strain (pretransformed with pKD46; encodes arabinose-inducible λ Red recombinase system) was used to seed 8.3 mL LB (¹/₅₀ dilution; ampicillin) and grown at 30 °C, ∼225 rpm, for 1 h. Arabinose was then added to 0.05% (∼3.3 mM; 20% stock), and incubation continued until OD₆₀₀ ≈ 0.4. After washing and concentrating cells to 50 μL in 20% glycerol, 200 to 400 ng of PCR product to be inserted (amplified from ∼100 pg template plasmid; purified, resuspended in water) was added for electroporation. Transformed cells were recovered for 2 h at 37 °C, ∼225 rpm, pelleted and plated on LB agar (with kanamycin, plus key metabolite to supplement the introduced auxotrophy), and incubated overnight at 37 °C. Typically ∼50 to 500 colonies were obtained, the large majority of which were successful integrants. It is worth noting that the integration of the PCR products (∼3.8 kbp) used in this work is pushing the known limits of the λ Red method. (49) In addition, thiamine pyrophosphate (50) knockouts (TPP; ΔthiL) were also attempted (TPP supplemented to 4.6 μg/mL (51, 52)), but for unknown reasons, we were unable to isolate an auxotroph.

Plasmid Stability Assay

Using 96-well plates, 0.2 μL of overnight culture under antibiotic selection pressure (ampicillin) was used to seed 200 μL of fresh LB (no antibiotic) and incubated at 37 °C, ∼850 rpm, for ∼6 h to achieve culture saturation (approximately 10 generations). Passaging was repeated until ∼100 generations were achieved, at which point 20 μL of a 1 × 10^–5 dilution was added to 50 μL fresh LB and plated on LB agar with or without antibiotic. Resultant colonies (from the nonselective plates) were examined for the presence of mRFP1 fluorescence as an indicator of plasmid retention using a hand-held 532 nm laser pointer in combination with the bandpass emission filter of a Visi-Blue transilluminator (Ultra-Violet Products Ltd.). For ColE2 CORs, where fluorescence in the low-copy DIAL strain AB was too faint to reliably assess, a combination of colony PCR, streak testing on selective media, and comparison of colony numbers on selective/nonselective media were instead made.

Soil Assay

One g of fresh or autoclaved garden soil (London, postcode SW10 0QP) was added to 3 mL of SOB medium (no antibiotic) and inoculated with 1 μL of overnight culture (grown in the presence of supplement where necessary). Cultures were grown overnight in 15 mL tubes at 30 °C, ∼225 rpm, and soil allowed to sediment before serial dilutions (1 × 10^–2 to 1 × 10^–7) were made using a 96-well plate (final volumes of 180 μL). Each dilution (10 μL) was dropped onto LB agar containing kanamycin to select for auxotrophic E. coli strain growth, allowed to dry, and incubated overnight at 30 °C. Only ColE2 CORs in medium copy (EI) DIAL strain auxotrophs were assessed; the wild-type control (i.e., prototroph) harbored an intermediary construction plasmid containing a R6K COR and kanR cassette.

Growth Curves

0.2 μL of overnight culture was diluted ¹/₉₀₀₀ in 1.8 mL LB (∼5 × 10⁴ colony forming units/mL (53)), with 200 μL subsequently aliquoted into a 96-well plate. Experiments were performed in situ using a POLARstar Omega microplate reader (BMG Labtech GMBH), at 37 °C, ∼700 rpm, with OD₆₀₀ read every 30 min. Data was collected using Omega (v1.02) and MARS Data Analysis software (v1.10; BMG Labtech GMBH), and growth curves manually shifted along the X-axis to compensate for any initial lag phase.

Western Blotting

500 μL aliquots of mid log phase cultures (¹/₁₀₀ overnight culture dilution, grown for 3 h at 37 °C, ∼225 rpm) were pelleted and resuspended in sufficient Laemmli sample buffer (54) to normalize OD₆₀₀ to 0.4 (in an assumed 50 μL volume), boiled for ∼2 min and then allowed to cool. For ζ-His/ε-His, 7 μL of prepared sample was loaded onto an Any kD Mini-PROTEAN TGX precast gel (Bio-Rad Laboratories Ltd.); for fainter Kid-His/Kis-His samples, 14 μL was used. The gel was run in a Mini-PROTEAN Tetra Cell (Bio-Rad Laboratories Ltd.) with 5 μL of PageRuler Prestained Protein Ladder (Fisher Scientific U.K. Ltd.), transferred to a PVDF membrane using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories Ltd.), and visualized with a WesternBreeze chromogenic immunodetection kit using Novex histidine tag (6×His) monoclonal mouse antibody (Life Technologies Ltd.) as the primary antibody at a concentration of 0.33 μg/mL (¹/₁₅₀₀ dilution) (all as per the manufacturer’s instructions).

Transformation Assay

For E. coli strains, 1 μL of 50 ng/μL plasmid stock was electroporated (1.8 kV, 0.1 cm electrocuvettes) into 50 μL aliquots of prepared cells (harvested mid log phase, washed and concentrated ∼133x in 20% glycerol, stored at −80 °C) using a MicroPulser electroporator (Bio-Rad Laboratories Ltd.) as per the manufacturer’s instructions, and recovered in 300 μL of LB for 1 h at 37 °C, ∼225 rpm. 100 μL was then plated on selective LB agar. For B. subtilis SCK6 (integrated P_xylA-comK (12)), 1 mL of overnight culture was diluted with 2 mL fresh LB, and 105 μL of filter-sterilized 30% d-xylose added to induce competence (1% final conc.). After 2 h at 37 °C, ∼225 rpm, 100 μL aliquots were taken, 5 μL of 20 ng/μL plasmid added and incubation continued for 90 min, after which all was plated on LB agar (with chloramphenicol). All plates were incubated overnight at 37 °C.

Biosensor Assay

Overnight culture (15 μL) was diluted ¹/₁₀₀ in 1.5 mL LB, with 180 μL subsequently added to wells containing 20 μL of serially diluted heavy-metal (32 μM to 0.5 μM Na₂HAsO₄; 6 to 0.5 μM HgCl₂; 2,000 to 31 μM CuSO₄) in a 96-well plate, mixing well. Sealed plates were incubated at 30 °C, ∼710 rpm, for 6 h prior to OD₆₀₀ and GFPmut3b fluorescence (485 nm excitation, 520 nm emission, with the gain set to 1000 and bottom reading optics used) being read on a POLARstar Omega microplate reader (BMG Labtech GMBH; see previous). Antibiotic (kanamycin) was only present in samples containing the original pSB3K3 plasmids.

Supporting Information

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Supplementary figures, sequence tables, GenBank accession numbers, and annotated plasmid ApE files (ApE can be downloaded for free at http://biologylabs.utah.edu/jorgensen/wayned/ape/). This material is available free of charge via the Internet at http://pubs.acs.org. In addition, selected GeneGuard plasmids may be obtained from the SEVA repository by request (see http://seva.cnb.csic.es/).

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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Corresponding Author
- Tom Ellis - Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, United Kingdom; Department of Bioengineering, Imperial College London, London SW7 2AZ, United Kingdom; Email: [email protected]
Authors
- Oliver Wright - Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, United Kingdom; Department of Bioengineering, Imperial College London, London SW7 2AZ, United Kingdom
- Mihails Delmans - Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, United Kingdom; Department of Bioengineering, Imperial College London, London SW7 2AZ, United Kingdom
- Guy-Bart Stan - Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, United Kingdom; Department of Bioengineering, Imperial College London, London SW7 2AZ, United Kingdom
Funding
This work was supported by a grant awarded through the Biotechnology and Biological Sciences Research Council (BBSRC)/Defence Science and Technology Laboratory (DSTL)/Engineering and Physical Sciences Research Council (EPSRC)/Medical Research Council (MRC) Joint Synthetic Biology Initiative (BB/J019720/1). G.B.S. and T.E. are further supported by EPSRC Science and Innovation Award EP/G036004/1.
Notes
The authors declare no competing financial interest.

Acknowledgment

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We wish to thank the Imperial College 2011 iGEM team “Auxin” for their initial work and ideas on a prototype GeneGuard system, and Richard Thomas (DSTL) for his comments throughout this research project. We also thank Prof J. Christopher Anderson (Dept of Bioengineering, University of California, Berkeley) for the gift of the DIAL strains and pBjk2992_jtk2828; (15) Prof Víctor de Lorenzo (Systems Biology Program, Centro Nacional de Biotecnología, Madrid) for the gift of SEVA plasmids; (10) Dr. Urszula Zielenkiewicz (Dept of Microbial Biochemistry, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences) for the gift of pBT286; (27) Prof. Ramón Díaz-Orejas (Dept of Molecular Microbiology and Infection Biology, Centro de Investigaciones Biológicas, Madrid) for the gift of pAB24; (55) and Dr. Baojun Wang (School of Biological Sciences, University of Edinburgh) for the gift of pSB3K3-Ars (aka pSB3K3-P_arsR-rbs30-gfp), pSB3K3-Mer (aka pSB3K3-P_merT-rbs32-gfp), and pSB3K3-Cus (aka pSB3K3-P_cusC-rbs31-gfp). (37)

References

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Rapid optimization of gene dosage in E. coli using DIAL strains
Kittleson, Joshua T.; Cheung, Sherine; Anderson, J. Christopher
Journal of Biological Engineering (2011), 5 (), 10CODEN: JBEOBZ; ISSN:1754-1611. (BioMed Central Ltd.)
Engineers frequently vary design parameters to optimize the behavior of a system. However, synthetic biologists lack the tools to rapidly explore a crit. design parameter, gene expression level, and have no means of systematically varying the dosage of an entire genetic circuit. As a step toward overcoming this shortfall, we have developed a technol. that enables the same plasmid to be maintained at different copy nos. in a set of closely related cells. This provides a rapid method for exploring gene or cassette dosage effects. We engineered two sets of strains to constitutively provide a trans-acting replication factor, either Pi of the R6K plasmid or RepA of the ColE2 plasmid, at different doses. Each DIAL (different allele) strain supports the replication of a corresponding plasmid at a const. level between 1 and 250 copies per cell. The plasmids exhibit cell-to-cell variability comparable to other popular replicons, but with improved stability. Since the origins are orthogonal, both replication factors can be incorporated into the same cell. We demonstrate the utility of these strains by rapidly assessing the optimal expression level of a model biosynthetic pathway for violecein. The DIAL strains can rapidly optimize single gene expression levels, help balance expression of functionally coupled genetic elements, improve investigation of gene and circuit dosage effects, and enable faster development of metabolic pathways.
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Durland, R. H. and Helinski, D. R. (1990) Replication of the broad-host-range plasmid RK2: Direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number J. Bacteriol. 172, 3849– 3858
[Crossref], [PubMed], [CAS], Google Scholar
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Replication of the broad-host-range plasmid RK2: direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number
Durland, Ross H.; Helinski, Donald R.
Journal of Bacteriology (1990), 172 (7), 3849-58CODEN: JOBAAY; ISSN:0021-9193.
The trfA gene of the broad-host-range plasmid RK2 is essential for initiation of plasmid replication. Two related TrfA proteins of 43 and 32 kDa are produced by independent translation initiation at two start codons within the trfA open reading frame. These proteins were overproduced in Escherichia coli and partially purified. Rabbit antisera raised against the 32-kDa TrfA protein (TrfA-32) and cross-reacting with the 43-kDa protein (TrfA-43) were used in Western blotting (immunoblotting) assays to measure intracellular TrfA levels. In logarithmically growing E. coli HB101, RK2 produced 4.6 ± 0.6 ng of TrfA-32 and 1.8 ± 0.2 ng of TrfA-43 per unit of optical d. at 600 nm (mean ± std. deviation). On the basis of detns. of the no. of cells per unit of optical d. at 600 nm, this corresponds to about 220 mols. of TrfA-32 and 80 mols. of TrfA-43 per cell. Dot blot hybridizations showed that plasmid RK2 is present in about 15 copies per E. coli cell under these conditions. Using plasmid constructs that produce different levels of TrfA proteins, the effect of excess TrfA on RK2 replication was tested. A two- to threefold excess of total TrfA increased the copy no. of RK2 by about 30%. Addnl. increases in TrfA protein concn. had no further effect on copy no., even at levels 170-fold above normal. An RK2 minimal origin plasmid showed a similar response to intracellular TrfA concn. These results demonstrate that TrfA protein concn. is not strictly rate limiting for RK2 replication and that a mechanism that is independent of TrfA concn. functions to limit RK2 copy no. in the presence of excess TrfA.
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Miki, T., Yasukochi, T., Nagatani, H., Furuno, M., Orita, T., Yamada, H., Imoto, T., and Horiuchi, T. (1987) Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: An application to overproduction of chicken lysozyme Protein Eng. 1, 327– 332
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Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: an application to overproduction of chicken lysozyme
Miki, Takeyoshi; Yasukochi, Takanori; Nagatani, Hiroko; Furuno, Masahiro; Orita, Tetsuro; Yamada, Hidenori; Imoto, Taiji; Horiuchi, Takao
Protein Engineering (1987), 1 (4), 327-32CODEN: PRENE9; ISSN:0269-2139.
A novel expression vector, pKP1500, for synthesizing unfused protein in E. coli was constructed. Plasmid pKP1500 perserves the tac promoter, the lacZ SD sequence, unique restriction sites (EcoRI, SmaI, BamHI, SalI, PstI and HindIII), and the rrnB terminators of pKK223-3, but the replication origin is replaced with that of pUC9. Construction of this plasmid is based upon that the copy no. control of pUC9 is temp. dependent. At 28°, the copy no. of pKP1500 is less than 25 per chromosome, approx. the same copy no. as that of pKK223-3, which contains the replication origin of pBR322, whereas at 42°, the copy no. increases about 10 times and reaches up to 230 copies per chromosome. The main advantage of this system is that the temp.-dependent copy control and regulatable expression of the tac promoter make cells carrying pKP1500 derivs. stable against selective pressure by detrimental overprodn. of foreign proteins at a low temp. and permits high expression of cloned DNAs at a high temp. When chicken lysozyme cDNA carrying the initiation codon (ATG) immediately upstream from the Lys1 codon was inserted downstream from the tac promoter and the SD sequence, the pKP1500 deriv. produced lysozyme at about 25% of the total cellular proteins. This value is more than 10 times higher than that obtained with the pKK223-3 deriv. carrying the same lysozyme cDNA. By comparison, the expression of eukaryotic genes from the tac promoter reported by others has usually been less than a few % of the total cellular protein. Plasmid pKP1500 would, therefore, be useful for the high level prodn. of unfused proteins from eukaryotic cDNAs in E. coli.
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Lin-Chao, S., Chen, W.-T., and Wong, T.-T. (1992) High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II Mol. Microbiol. 6, 3385– 3393
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High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II
Lin-Chao, Sue; Chen, Wen Tsuan; Wong, Ten Tsao
Molecular Microbiology (1992), 6 (22), 3385-93CODEN: MOMIEE; ISSN:0950-382X.
The plasmids pUC18 and pUC19 are pBR322 derivs. that replicate at a copy no. several fold higher than the parent during growth of Escherichia coli at 37°. The authors show here that the high copy no. of pUC plasmids results from a single point mutation in the replication primer, RNA II, and that the phenotypic effects of this mutation can be suppressed by the Rom (RNA one modulator)/Rop protein or by lowering the growth temp. to 30°. The mutation's effects are enhanced by cell growth at 42°, at which copy no. is further increased. During normal cell growth, the pUC mutation does not affect the length or function of RNA I, the antisense repressor of plasmid DNA replication, but may, as computer anal. suggests, alter the secondary structure of pUC RNA II. The authors suggest that the pUC mutation impedes interactions between the repressor and the primer by producing a temp.-dependent alteration of the RNA II conformation. The Rom/Rop protein may either promote normal folding of the mutated RNA II or, alternatively, may enable the interaction of sub-optimally folded RNA II with the repressor.
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Wong, Q. N. Y., Ng, V. C. W., Lin, M. C. M., Kung, H.-F., Chan, D., and Huang, J.-D. (2005) Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli Nucleic Acids Res. 33, e59
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Acord, J. and Masters, M. (2004) Expression from the Escherichia coli dapA promoter is regulated by intracellular levels of diaminopimelic acid FEMS Microbiol. Lett. 235, 131– 137
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There is no corresponding record for this reference.
21
Datsenko, K. A. and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products Proc. Natl. Acad. Sci. U.S.A. 97, 6640– 6645
[Crossref], [PubMed], [CAS], Google Scholar
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One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
Datsenko, Kirill A.; Wanner, Barry L.
Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (12), 6640-6645CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
The authors have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homol. to the targeted gene(s). In this procedure, recombination requires the phage λ Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy no. plasmid. To demonstrate the utility of this approach, the authors generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the resp. PCR products, the authors made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, esp. in genome anal. of E. coli and other bacteria because the procedure can be done in wild-type cells.
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Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K. A., Tomita, M., Wanner, B. L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection Mol. Syst. Biol. 2, 0008
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Shetty, R. P., Endy, D., and Knight, T. F. (2008) Engineering BioBrick vectors from BioBrick parts J. Biol. Eng. 2, 5
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Engineering BioBrick vectors from BioBrick parts
Shetty Reshma P; Endy Drew; Knight Thomas F Jr
Journal of biological engineering (2008), 2 (), 5 ISSN:.
BACKGROUND: The underlying goal of synthetic biology is to make the process of engineering biological systems easier. Recent work has focused on defining and developing standard biological parts. The technical standard that has gained the most traction in the synthetic biology community is the BioBrick standard for physical composition of genetic parts. Parts that conform to the BioBrick assembly standard are BioBrick standard biological parts. To date, over 2,000 BioBrick parts have been contributed to, and are available from, the Registry of Standard Biological Parts. RESULTS: Here we extended the same advantages of BioBrick standard biological parts to the plasmid-based vectors that are used to provide and propagate BioBrick parts. We developed a process for engineering BioBrick vectors from BioBrick parts. We designed a new set of BioBrick parts that encode many useful vector functions. We combined the new parts to make a BioBrick base vector that facilitates BioBrick vector construction. We demonstrated the utility of the process by constructing seven new BioBrick vectors. We also successfully used the resulting vectors to assemble and propagate other BioBrick standard biological parts. CONCLUSION: We extended the principles of part reuse and standardization to BioBrick vectors. As a result, myriad new BioBrick vectors can be readily produced from all existing and newly designed BioBrick parts. We invite the synthetic biology community to (1) use the process to make and share new BioBrick vectors; (2) expand the current collection of BioBrick vector parts; and (3) characterize and improve the available collection of BioBrick vector parts.
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Silby, M. W. and Levy, S. B. (2004) Use of in vivo expression technology to identify genes important in growth and survival of Pseudomonas fluorescens Pf0-1 in soil: Discovery of expressed sequences with novel genetic organization J. Bacteriol. 186, 7411– 7419
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Use of in vivo expression technology to identify genes important in growth and survival of Pseudomonas fluorescens Pf0-1 in soil: Discovery of expressed sequences with novel genetic organization
Silby, Mark W.; Levy, Stuart B.
Journal of Bacteriology (2004), 186 (21), 7411-7419CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
Studies were undertaken to det. the genetic needs for the survival of Pseudomonas fluorescens Pf0-1, a gram-neg. soil bacterium potentially important for biocontrol and bioremediation, in soil. In vivo expression technol. (IVET) identified 22 genes with elevated expression in soil relative to lab. media. Soil-induced sequences included genes with probable functions of nutrient acquisition and use, and of gene regulation. Ten sequences, lacking similarity to known genes, overlapped divergent known genes, revealing a novel genetic organization at those soil-induced loci. Mutations in three soil-induced genes led to impaired early growth in soil but had no impact on growth in lab. media. Thus, IVET studies have identified sequences important for soil growth and have revealed a gene organization that was undetected by traditional lab. approaches.
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Varivarn, K., Champa, L. A., Silby, M. W., and Robleto, E. A. (2013) Colonization strategies of Pseudomonas fluorescens Pf0-1: Activation of soil-specific genes important for diverse and specific environments BMC Microbiol. 13, 92
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Colonization strategies of Pseudomonas fluorescens Pf0-1: activation of soil-specific genes important for diverse and specific environments
Varivarn, Katila; Champa, Lindsey A.; Silby, Mark W.; Robleto, Eduardo A.
BMC Microbiology (2013), 13 (), 92CODEN: BMMIBC; ISSN:1471-2180. (BioMed Central Ltd.)
Background: Pseudomonas fluorescens is a common inhabitant of soil and the rhizosphere environment. In addn. to potential applications in biocontrol and bioremediation, P. fluorescens is of interest as a model for studying bacterial survival and fitness in soil. A previous study using in vivo expression technol. (IVET) identified 22 genes in P. fluorescens Pf0-1 which are up-regulated during growth in Massachusetts loam soil, a subset of which are important for fitness in soil. Despite this and other information on adaptation to soil, downstream applications such as biocontrol or bioremediation in diverse soils remain underdeveloped. We undertook an IVET screen to identify Pf0-1 genes induced during growth in arid Nevada desert soil, to expand our understanding of growth in soil environments, and examine whether Pf0-1 uses general or soil type-specific mechanisms for success in soil environments. Results: Twenty six genes were identified. Consistent with previous studies, these genes cluster in metab., information storage/processing, regulation, and 'hypothetical', but there was no overlap with Pf0-1 genes induced during growth in loam soil. Mutation of both a putative glutamine synthetase gene (Pfl01_2143) and a gene predicted to specify a component of a type VI secretion system (Pfl01_5595) resulted in a decline in arid soil persistence. When examd. in sterile loam soil, mutation of Pfl01_5595 had no discernible impact. In contrast, the Pfl01_2143 mutant was not impaired in persistence in sterile soil, but showed a significant redn. in competitive fitness. Conclusions: These data support the conclusion that numerous genes are specifically important for survival and fitness in natural environments, and will only be identified using in vivo approaches. Furthermore, we suggest that a subset of soil-induced genes is generally important in different soils, while others may contribute to success in specific types of soil. The importance of glutamine synthetase highlights a crit. role for nitrogen metab. in soil fitness. The implication of Type 6 secretion underscores the importance of microbial interactions in natural environments. Understanding the general and soil-specific genes will greatly improve the persistence of designed biocontrol and bioremediation strains within the target environment.
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Molin, S., Boe, L., Jensen, L. B., Kristensen, C. S., Givskov, M., Ramos, J. L., and Bej, A. K. (1993) Suicidal genetic elements and their use in biological containment of bacteria Annu. Rev. Microbiol. 47, 139– 166
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Suicidal genetic elements and their use in biological containment of bacteria
Molin, S.; Boe, L.; Jensen, L. B.; Kristensen, C. S.; Givskov, M.; Ramos, J. L.; Bej, A. K.
Annual Review of Microbiology (1993), 47 (), 139-66CODEN: ARMIAZ; ISSN:0066-4227.
A review with 70 refs.
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Zielenkiewicz, U. and Ceglowski, P. (2005) The toxin–antitoxin system of the streptococcal plasmid pSM19035 J. Bacteriol. 187, 6094– 6105
[Crossref], [PubMed], [CAS], Google Scholar
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The toxin-antitoxin system of the streptococcal plasmid pSM19035
Zielenkiewicz, Urszula; Ceglowski, Piotr
Journal of Bacteriology (2005), 187 (17), 6094-6105CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
PSM19035 of the pathogenic bacterium Streptococcus pyogenes is a low-copy-no. plasmid carrying erythromycin resistance, stably maintained in a broad range of gram-pos. bacteria. We show here that the ω-ε-ζ operon of this plasmid constitutes a novel protein plasmid addiction system in which the ε and ζ genes encode an antitoxin and toxin, resp., while ω plays an autoregulatory function. Expression of toxin Zeta is bactericidal for the gram-pos. Bacillus subtilis and bacteriostatic for the gram-neg. Escherichia coli. The toxic effects of ζ gene expression in both bacterial species are counteracted by proper expression of ε. The ε-ζ toxin-antitoxin cassette stabilizes plasmids in E. coli less efficiently than in B. subtilis.
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Zielenkiewicz, U., Kowalewska, M., Kaczor, C., and Ceglowski, P. (2009) In vivo interactions between toxin–antitoxin proteins epsilon and zeta of streptococcal plasmid pSM19035 in Saccharomyces cerevisiae J. Bacteriol. 191, 3677– 3684
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In vivo interactions between toxin-antitoxin proteins Epsilon and Zeta of streptococcal plasmid pSM19035 in Saccharomyces cerevisiae
Zielenkiewicz, Urszula; Kowalewska, Magdalena; Kaczor, Celina; Ceglowski, Piotr
Journal of Bacteriology (2009), 191 (11), 3677-3684CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
The widespread prokaryotic toxin-antitoxin (TA) systems involve conditional interaction between two TA proteins. The interaction between the Epsilon and Zeta proteins, constituting the TA system of plasmid pSM19035 from Streptococcus pyogenes, was detected in vivo using a yeast two-hybrid system. As we showed using Saccharomyces cerevisiae, the Zeta toxin hybrid gene also exerts its toxic effects in a dose-dependent manner in eukaryotic cells. Anal. of mutant proteins in the two-hybrid system demonstrated that the N-terminal part of Zeta and the N-terminal region of Epsilon are involved in the interaction. The N-terminal region of the Zeta protein and its ATP/GTP binding motif were found to be responsible for the toxicity.
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de la Cueva-Méndez, G., Mills, A. D., Clay-Farrace, L., Díaz-Orejas, R., and Laskey, R. A. (2003) Regulatable killing of eukaryotic cells by the prokaryotic proteins Kid and Kis EMBO J. 22, 246– 251
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Mutschler, H., Gebhardt, M., Shoeman, R. L., and Meinhart, A. (2011) A novel mechanism of programmed cell death in bacteria by toxin–antitoxin systems corrupts peptidoglycan synthesis PLoS Biol. 9, e1001033
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Pimentel, B., Madine, M. A., and de la Cueva-Méndez, G. (2005) Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1 EMBO J. 24, 3459– 3469
[Crossref], Google Scholar
There is no corresponding record for this reference.
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Salis, H. M., Mirsky, E. A., and Voigt, C. A. (2009) Automated design of synthetic ribosome binding sites to control protein expression Nat. Biotechnol. 27, 946– 950
[Crossref], [PubMed], [CAS], Google Scholar
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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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Casini, A., MacDonald, J. T., Jonghe, J. D., Christodoulou, G., Freemont, P. S., Baldwin, G. S., and Ellis, T. (2013) One-pot DNA construction for synthetic biology: The Modular Overlap-Directed Assembly with Linkers (MODAL) strategy Nucleic Acids Res. 42, e7
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de la Cueva-Méndez, G. and Pimentel, B. (2007) Gene and cell survival: Lessons from prokaryotic plasmid R1 EMBO Rep. 8, 458– 464
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Olson, D. G. and Lynd, L. R. (2012) Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles J. Biol. Eng. 6, 5
[Crossref], [PubMed], [CAS], Google Scholar
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Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles
Olson, Daniel G.; Lynd, Lee R.
Journal of Biological Engineering (2012), 6 (), 5CODEN: JBEOBZ; ISSN:1754-1611. (BioMed Central Ltd.)
Background: Temp.-sensitive (Ts) plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram pos., thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. for predicting Ts mutants based on the amino-acid sequence of the protein. Results: A library of 34 mutant plasmids was designed, synthesized and screened, resulting in 6 mutants which exhibited a Ts phenotype. Of these 6, the one with the most temp.-sensitive phenotype (M166A) was compared with the original plasmid. It exhibited lower stability at 48°C and was completely unable to replicate at 55°C. Conclusions: The plasmid described in this work could be useful in future efforts to genetically engineer C. thermocellum and the method used to generate this plasmid may be useful for others trying to make Ts plasmids.
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Pellegrini, O., Mathy, N., Gogos, A., Shapiro, L., and Condon, C. (2005) The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor Mol. Microbiol. 56, 1139– 1148
[Crossref], [PubMed], [CAS], Google Scholar
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The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor
Pellegrini, Olivier; Mathy, Nathalie; Gogos, Arhonda; Shapiro, Lawrence; Condon, Ciaran
Molecular Microbiology (2005), 56 (5), 1139-1148CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
Operons encoding stable toxins and their labile antidote are widespread in prokaryotes and play important roles in plasmid partitioning and cellular responses to stress. One such family of toxins MazF/ChpAK/PemK encodes an endoribonuclease that inactivates cellular mRNAs by cleaving them at specific, but frequently occurring sites. Here the authors show that the Bacillus subtilis ydcE gene encodes a member of this family of RNases, which the authors have called EndoA. Overexpression of EndoA is toxic for bacterial cell growth and this toxicity is reversed by coexpression of the gene immediately upstream, ydcD. Furthermore, YdcD inhibits EndoA activity directly in vitro. EndoA has similar cleavage specificity to MazF and PemK and yields cleavage products with 3'-phosphate and 5'-hydroxyl groups, typical of EDTA-resistant degradative RNases. This is the first example of an antitoxin-toxin system in B. subtilis.
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Wang, B., Barahona, M., and Buck, M. (2013) A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals Biosens. Bioelectron. 40, 368– 376
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Friedland, A. E., Lu, T. K., Wang, X., Shi, D., Church, G., and Collins, J. J. (2009) Synthetic gene networks that count Science 324, 1199– 1202
[Crossref], [PubMed], [CAS], Google Scholar
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Synthetic Gene Networks That Count
Friedland, Ari E.; Lu, Timothy K.; Wang, Xiao; Shi, David; Church, George; Collins, James J.
Science (Washington, DC, United States) (2009), 324 (5931), 1199-1202CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one the ability to program and design cells with some of the principles of modern computing, such as counting. A cellular counter would enable complex synthetic programming and a variety of biotechnol. applications. Here, we report two complementary synthetic genetic counters in Escherichia coli that can count up to three induction events: the first, a riboregulated transcriptional cascade, and the second, a recombinase-based cascade of memory units. These modular devices permit counting of varied user-defined inputs over a range of frequencies and can be expanded to count higher nos.
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Callura, J. M., Dwyer, D. J., Isaacs, F. J., Cantor, C. R., and Collins, J. J. (2010) Tracking, tuning, and terminating microbial physiology using synthetic riboregulators Proc. Natl. Acad. Sci. U.S.A. 107, 15898– 15903
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Ronchel, M. C. and Ramos, J. L. (2001) Dual system to reinforce biological containment of recombinant bacteria designed for rhizoremediation Appl. Environ. Microbiol. 67, 2649– 2656
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Marris, C. and Jefferson, C. (2013) Synthetic Biology: Containment and Release of Engineered Micro-organisms. Workshop held at King’s College London, 29th April 2013. Summary of discussions available from http://www.kcl.ac.uk/sspp/departments/sshm/research/Research-Labs/CSynBi-Events.aspx.
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World Health Organization. ( (2011) Guidelines for Drinking-Water Quality, 4th ed., WHO Press, Geneva. Available from http://www.who.int/.
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Kang, H. Y., Dozois, C. M., Tinge, S. A., Lee, T. H., and Curtiss, R., III (2002) Transduction-mediated transfer of unmarked deletion and point mutations through use of counterselectable suicide vectors J. Bacteriol. 184, 307– 312
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Transduction-mediated transfer of unmarked deletion and point mutations through use of counterselectable suicide vectors
Kang, Ho Young; Dozois, Charles M.; Tinge, Steven A.; Lee, Tae Ho; Curtiss, Roy, III
Journal of Bacteriology (2002), 184 (1), 307-312CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
A challenge in strain construction is that unmarked deletion and nucleotide substitution alleles generally do not confer selectable phenotypes. We describe here a rapid and efficient strategy for transferring such alleles via generalized transduction. The desired allele is first constructed and introduced into the chromosome by conventional allelic-exchange methods. The suicide vector contg. the same allele is then integrated into the mutant chromosome, generating a tandem duplication homozygous for that allele. The resulting strain is used as a donor for transductional crosses, and selection is made for a marker carried by the integrated suicide vector. Segregation of the tandem duplication results in haploid individuals, each of which carries the desired allele. To demonstrate this mutagenesis strategy, we used bacteriophage P22HTint for generalized transduction-mediated introduction of unmarked mutations to Salmonella enterica serovar Typhimurium. This method is applicable to any species for which generalized transduction is established.
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Sat, B., Reches, M., and Engelberg-Kulka, H. (2003) The Escherichia coli mazEF suicide module mediates thymineless death J. Bacteriol. 185, 1803– 1807
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The Escherichia coli mazEF suicide module mediates thymineless death
Sat, Boaz; Reches, Myriam; Engelberg-Kulka, Hanna
Journal of Bacteriology (2003), 185 (6), 1803-1807CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
In 1954, Cohen and Barner discovered that a thymine auxotrophic (thyA) mutant of Escherichia coli undergoes cell death in response to thymine starvation. This phenomenon, called thymineless death (TLD), has also been found in many other organisms, including prokaryotes and eukaryotes. Though TLD has been studied intensively, its mol. mechanism has not yet been explained. Previously we reported on the E. coli mazEF system, a regulatable chromosomal suicide module that can be triggered by various stress conditions. MazF is a stable toxin, and MazE is an unstable antitoxin. Here, we show that cell death that is mediated by the mazEF module can also be activated by thymine starvation. We found that TLD depends on E. coli mazEF and that under thymine starvation, the activity of the mazEF promoter P2 is significantly reduced. Our results, which describe thymine starvation as a trigger for a built-in death program, have implications for programmed cell death in both prokaryotes and eukaryotes.
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Steidler, L., Neirynck, S., Huyghebaert, N., Snoeck, V., Vermeire, A., Goddeeris, B., Cox, E., Remon, J. P., and Remaut, E. (2003) Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10 Nat. Biotechnol. 21, 785– 789
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Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10
Steidler, Lothar; Neirynck, Sabine; Huyghebaert, Nathalie; Snoeck, Veerle; Vermeire, An; Goddeeris, Bruno; Cox, Eric; Remon, Jean Paul; Remaut, Erik
Nature Biotechnology (2003), 21 (7), 785-789CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease. However, the release of such genetically modified organisms through clin. use raises safety concerns. In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene. This thyA- hIL10+ L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biol. containment system and the bacterium's capacity to secrete hIL-10 were validated in vivo in pigs. Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strain acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.
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Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., and Mattick, J. S. (1991) Touchdown PCR to circumvent spurious priming during gene amplification Nucleic Acids Res. 19, 4008
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Stemmer, W. P., Crameri, A., Ha, K. D., Brennan, T. M., and Heyneker, H. L. (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides Gene 164, 49– 53
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Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides
Stemmer, Willem P. C.; Crameri, Andreas; Ha, Kim D.; Brennan, Thomas M.; Heyneker, Herbert L.
Gene (1995), 164 (1), 49-53CODEN: GENED6; ISSN:0378-1119. (Elsevier)
Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large nos. of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling (Stemmer, W.P.C. 1994), does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment contg. the TEM-1 β-lactamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene was PCR amplified and cloned in a vector contg. the tetracycline-resistance gene (TcR) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the TcR colonies were ApR, making this approach a general method for the rapid and cost-effective synthesis of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel contg. 134 oligos, a high-mol.-mass multimeric form of a 2.7-kb plasmid contg. the bla gene, the α-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.
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Kuhlman, T. E. and Cox, E. C. (2010) Site-specific chromosomal integration of large synthetic constructs Nucleic Acids Res. 38, e92
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Site-specific chromosomal integration of large synthetic constructs
Kuhlman, Thomas E.; Cox, Edward C.
Nucleic Acids Research (2010), 38 (6), e92/1-e92/10CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
The authors have developed an effective, easy-to-use two-step system for the site-directed insertion of large genetic constructs into arbitrary positions in the Escherichia coli chromosome. The system uses λ-Red mediated recombineering accompanied by the introduction of double-strand DNA breaks in the chromosome and a donor plasmid bearing the desired insertion fragment. This method, in contrast to existing recombineering or phage-derived insertion methods, allows for the insertion of very large fragments into any desired location and in any orientation. This method was demonstrated by inserting a 7-kb fragment consisting of a venus-tagged lac repressor gene along with a target lacZ reporter into six unique sites distributed sym. about the chromosome. The universality and repeatability of the method was shown by sep. inserting the lac repressor gene and the lacZ target into the chromosome at sep. locations around the chromosome via repeated application of the protocol.
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thiK and thiL loci of Escherichia coli
Imamura, Nariko; Nakayama, Hideo
Journal of Bacteriology (1982), 151 (2), 708-17CODEN: JOBAAY; ISSN:0021-9193.
Mutants of E. coli K-12 auxotrophic for thiamin phosphates were produced in stepwise fashion from the polyauxotrophic F- strain JC1552, via intermediate prodn. of thiamin auxotrophs that had lost the enzymic activity of either phosphomethylpyrimidine kinase or thiamin phosphate pyrophosphorylase. They include 2 types: one responds to thiamin monophosphate or thiamin pyrophosphate, and the other responds to thiamin pyrophosphate only; the former lacks thiamin kinase activity, and the latter lacks thiamin monophosphate kinase activity, in addn. to the enzymic defects caused by the 1st mutations. Two new genes were found for which the designations thiK and thiL are proposed, which govern the activities of thiamin kinase and thiamin monophosphate kinase, resp. By conjugation and P1 transduction, the thiK locus was mapped at ∼25 min, between pyrC and purB and close to fabD. The relative order of thiK with respect to nearby genes was tentatively established as pyrC-pstG-fabD-thiK-purB. In the case of thiL, the locus was situated at ∼9 min, between tsx and acrA and probably 0.2 min clockwise from the former.
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Webb, E. and Downs, D. (1997) Characterization of thiL, encoding thiamin-monophosphate kinase Salmonella typhimurium J. Biol. Chem. 272, 15702– 15707
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Schyns, G., Potot, S., Geng, Y., Barbosa, T. M., Henriques, A., and Perkins, J. B. (2005) Isolation and characterization of new thiamine-deregulated mutants of Bacillus subtilis J. Bacteriol. 187, 8127– 8136
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Isolation and characterization of new thiamine-deregulated mutants of Bacillus subtilis
Schyns, Ghislain; Potot, Sebastien; Geng, Yi; Barbosa, Teresa M.; Henriques, Adriano; Perkins, John B.
Journal of Bacteriology (2005), 187 (23), 8127-8136CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
In bacteria, thiamin pyrophosphate (TPP) is an essential cofactor that is synthesized de novo. Thiamin, however, is not an intermediate in the biosynthetic pathway but is salvaged from the environment and phosphorylated to TPP. We have isolated and characterized new mutants of Bacillus subtilis that deregulate thiamin biosynthesis and affect the export of thiamin products from the cell. Deletion of the ydiA gene, which shows significant similarity to the thiamin monophosphate kinase gene of Escherichia coli (thiL), did not generate the expected thiamin auxotroph but instead generated a thiamin bradytroph that grew to near-wild-type levels on minimal medium. From this ΔthiL deletion mutant, two addnl. Et methanesulfonate-induced mutants that derepressed the expression of a thiC-lacZ transcriptional reporter were isolated. One mutant, Tx1, contained a nonsense mutation within the B. subtilis yloS (thiN) gene that encodes a thiamin pyrophosphokinase, a result which confirmed that B. subtilis contains a single-step, yeast-like thiamin-to-TPP pathway in addn. to the bacterial TPP de novo pathway. A second mutant, strain Tx26, was shown to contain two lesions. Genetic mapping and DNA sequencing indicated that the first mutation affected yuaJ, which encodes a thiamin permease. The second mutation was located within the ykoD cistron of the ykoFEDC operon, which putatively encodes the ATPase component of a unique thiamin-related ABC transporter. Genetic and microarray studies indicated that both the mutant yuaJ and ykoD genes were required for the derepression of thiamin-regulated genes. Moreover, the combination of the four mutations (the ΔthiL, thiN, yuaJ, and ykoD mutations) into a single strain significantly increased the prodn. and excretion of thiamin products into the culture medium. These results are consistent with the proposed "riboswitch" mechanism of thiamin gene regulation.
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Domínguez, M. C., La Rosa, de M., and Borobio, M. V. (2001) Application of a spectrophotometric method for the determination of post-antibiotic effect and comparison with viable counts in agar J. Antimicrob. Chemother. 47, 391– 398
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Application of a spectrophotometric method for the determination of post-antibiotic effect and comparison with viable counts in agar
Dominguez, M. Carmen; De la Rosa, Manuel; Borobio, M. Victoria
Journal of Antimicrobial Chemotherapy (2001), 47 (4), 391-398CODEN: JACHDX; ISSN:0305-7453. (Oxford University Press)
The post-antibiotic effects of gentamicin and ciprofloxacin at 1 ×, 2 × and 4 × MIC on Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 were studied using a spectrophotometric method and the classic method of viable counts on agar as a ref. Monitoring of the growth kinetics was carried out by viability counting on the plate every hour and by means of the optical d. of the cultures measured by spectrophotometry at a wavelength of 450 nm. No statistically significant differences were found between the results obtained with the spectrophotometric method and the ref. method. The former method was much quicker, much easier to use and to replicate.
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Cleavage of structural proteins during the assembly of the head of bacteriophage T4
Laemmli, U. K.
Nature (London, United Kingdom) (1970), 227 (5259), 680-685CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
Using an improved method of polyacrylamide gel electrophoresis based on the capability of SDS to break down proteins into their individual polypeptide chains, many previously unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
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Bravo, A., Torrontegui, G., and Díaz, R. (1987) Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid Mol. Gen. Genet. 210, 101– 110
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Identification of components of a new stability system of plasmid R1, ParD, that is close to to the origin of replication of this plasmid
Bravo, Alicia; De Torrontegui, Gertrudis; Diaz, Ramon
Molecular and General Genetics (1987), 210 (1), 101-10CODEN: MGGEAE; ISSN:0026-8925.
A mutation which derepresses an autoregulated system that is located in the vicinity of the basic replicon of R1 stabilizes the ParA- and ParB- miniplasmid of R1, pKN1562, without increasing its copy no. The system, called ParD, maps inside the 1.45-kb PstI-EcoRI fragment that is adjacent to the origin of replication of the plasmid. Two proteins whose expression is coordinated are components of the system. The sequence of the PstI-EcoRI fragment was obtained. The wild-type ParD system dets. in cis a basal but detectable stability.
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Figure 1
Figure 1. Flow cytometry of DIAL strains hosting COR reporter plasmids. (a) Schematic of COR plasmid dependence on host DIAL strain, where plasmid copy number is tuned by the ribosome binding site (RBS) strength of the replication initiator protein transcript. (b) DIAL strains constitutively expressing both π and RepA at low (AB), medium (EI) and high (JK) levels were transformed with mRFP1 reporter plasmids containing the R6K (pSEVA117Rb) or ColE2 (pSEVA177Rb) COR, and fluorescence assessed at mid log growth phase by flow cytometry. Low-copy RK2 (pSEVA127Rb) and high-copy pUC (pSEVA167Rb) origins were also profiled as controls. Median fluorescence values and robust coefficient of variation (rCV) are indicated beneath each plot (n = 4 biological repeats; representatives shown). W/T, wild-type E. coli MC1061 used for DIAL strain construction; au, arbitrary units; X-axis, side scatter; RBS EI, RBS E for π, RBS I for RepA (see Supporting Information Table 1 for more detail).
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Figure 2
Figure 2. Plasmid stability in auxotrophic strains. (a) Schematic of DIAL strain auxotroph (ΔthyA or ΔdapA) dependence on complementing plasmid (thyA⁺ or dapA⁺). Orange arrow indicates complementation of knocked-out gene (remnants represented by orange bars on chromosome). (b) Stability assay measuring the proportion of colonies that retain their plasmid in low-copy DIAL strain AB after ∼100 generations in liquid LB without antibiotic selection (n = 4 biological repeats; error bars = standard deviation). (c) Assay as above, but plated on agar containing key metabolite to assess plasmid stability when auxotrophic pressure is removed (n = 4 biological repeats; error bars = standard deviation). (d) Assessment nonsterile soil’s ability to provide key metabolite to auxotrophs when added to liquid SOB and incubated overnight (dilution series subsequently plated on kanamycin-containing LB agar to suppress growth of other soil microbes). kanR, kanamycin resistance cassette; dark purple bars, FRT (flippase recognition target); thyd., thymidine; DAP, diaminopimelic acid; KO, knockout; R6K (pSEVA117Rb), ColE2 (pSEVA177Rb), R6K-thyA (pSEVA117RbT), ColE2-thyA (pSEVA177RbT), R6K-dapA (pSEVA117RbD), ColE2-dapA (pSEVA177RbD), low RK2 (pSEVA127Rb), high pUC (pSEVA167Rb) (see Supporting Information Table 1 for more detail).
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Figure 3
Figure 3. Use of ζ–ε and Kid-Kis toxin–antitoxin pairs. (a) Schematic of how a toxin-encoding plasmid may prove deleterious if taken up by wild-type cells, while the specified host cell possesses genome-encoded immunity. (b) Growth curves of high-copy DIAL strain JK with various combinations of ΔdapA auxotrophy, chromosomally integrated ε or Kis antitoxin, and plasmid-encoded ζ or Kid toxin (ColE2 COR used; n = 3 biological repeats; error bars = standard deviation). For ΔthyA auxotrophy, and other plasmid copy numbers, see Supporting Information Figure 2. (c) Western blot of various DIAL strains constitutively expressing integrated ε (11.5 kDa) or Kis (10.2 kDa) antitoxins alone, as well as with plasmid-encoded ζ (33.2 kDa) or Kid (12.7 kDa) toxins. All toxins/antitoxins are His-tagged at the C-terminus; putative toxin bands are arrowed. (d) Transformation assessment of ability of wild-type cells to maintain toxin plasmid in the absence of integrated antitoxin. W/T, wild-type; antiT, antitoxin; WM, weight marker; dapA (pSEVA177RbD); dapA-ζ (pSEVA177RbDZh); dapA-Kid (pSEVA177RbDKh); control plasmid (pSEVA3b61); ζ plasmid (pSEVA3b6Zh); Kid plasmid (pSEVA3b6Kh).
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Figure 4
Figure 4. Schematic of the GeneGuard system. The genomic cassette (a) consists of replication initiator, antitoxin and FRT-bound kanamycin resistance genes, flanked by ∼280 to 500 bp of 5′/3′ UTR sequence from the thyA or dapA genes (total cassette size of ∼3.6–3.8 kbp). The vector cassette (b) hosts cargo DNA via a pUC18-derived multicloning site that contains a removable antibiotic resistance gene between the PacI and AvrII sites. To retrofit existing plasmids, the COR/toxin/auxotrophy cassette (c) may be PCR-amplified and swapped with the existing origin/antibiotic resistance region. After construction, GeneGuard-derived plasmids are dependent on host cells that contain the requisite genomic cassette (d).
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Figure 5
Figure 5. GeneGuard system applied to heavy-metal biosensors. (a) Schematic of biosensors inserted into GeneGuard plasmids. Arsenic relieves ArsR repression of ParsR; mercury relieves MerR repression of PmerT; and copper enables CusR activation of PcusC. Each of these promoters is linked to the reporter GFPmut3b. (b) Dose response curves in E. coli DH10B for the arsenic biosensor in its original plasmid (pSB3K3 contains a medium-copy p15A origin, requires kanamycin selection), and its performance when ported to GeneGuard variants pD1K (dapA, R6K COR, Kid toxin; no antibiotic selection used) and pT7Z (thyA, ColE2 COR, ζ toxin; no antibiotic selection used) with the requisite genomic cassettes inserted to support a medium-copy plasmid number (n = 4 biological repeats; error bars = standard deviation). (c) Dose response curves for the mercury biosensor, as per part b. (d) Dose response curves for the copper biosensor, as per part b. W/T, wild-type; au, arbitrary units; WHO, World Health Organization. (43) For low and high-copy GeneGuard plasmid results, see Supporting Information Figure 3.
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  8
  Xenobiology: a new form of life as the ultimate biosafety tool
  Schmidt, Markus
  BioEssays (2010), 32 (4), 322-331CODEN: BIOEEJ; ISSN:0265-9247. (Wiley-Blackwell)
  A review. Synthetic biologists try to engineer useful biol. systems that do not exist in nature. One of their goals is to design an orthogonal chromosome different from DNA and RNA, termed XNA for xeno nucleic acids. XNA exhibits a variety of structural chem. changes relative to its natural counterparts. These changes make this novel information-storing biopolymer "invisible" to natural biol. systems. The lack of cognition to the natural world, however, is seen as an opportunity to implement a genetic firewall that impedes exchange of genetic information with the natural world, which means it could be the ultimate biosafety tool. Here I discuss, why it is necessary to go ahead designing xenobiol. systems like XNA and its XNA binding proteins; what the biosafety specifications should look like for this genetic enclave; which steps should be carried out to boot up the first XNA life form; and what it means for the society at large.
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9. 9
  Benner, S. A. and Sismour, A. M. (2005) Synthetic biology Nat. Rev. Genet. 6, 533– 543
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  9
  Synthetic biology
  Benner, Steven A.; Sismour, A. Michael
  Nature Reviews Genetics (2005), 6 (7), 533-543CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)
  A review. Synthetic biologists come in two broad classes. One uses unnatural mols. to reproduce emergent behaviors from natural biol., with the goal of creating artificial life. The other seeks interchangeable parts from natural biol. to assemble into systems that function unnaturally. Either way, a synthetic goal forces scientists to cross uncharted ground to encounter and solve problems that are not easily encountered through anal. This drives the emergence of new paradigms in ways that anal. cannot easily do. Synthetic biol. has generated diagnostic tools that improve the care of patients with infectious diseases, as well as devices that oscillate, creep and play tic-tac-toe.
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10. 10
  Silva-Rocha, R., Martínez-García, E., Calles, B., Chavarría, M., Arce-Rodríguez, A., Las de Heras, A., Páez-Espino, A. D., Durante-Rodríguez, G., Kim, J., Nikel, P. I., Platero, R., and de Lorenzo, V. (2012) The Standard European Vector Architecture (SEVA): A coherent platform for the analysis and deployment of complex prokaryotic phenotypes Nucleic Acids Res. 41, D666– D675
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  There is no corresponding record for this reference.
11. 11
  Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q.-A., Tran, A. B., Paull, M., Keasling, J. D., Arkin, A. P., and Endy, D. (2013) Precise and reliable gene expression via standard transcription and translation initiation elements Nat. Methods 10, 354– 360
  [Crossref], [PubMed], [CAS], Google Scholar
  11
  Precise and reliable gene expression via standard transcription and translation initiation elements
  Mutalik, Vivek K.; Guimaraes, Joao C.; Cambray, Guillaume; Lam, Colin; Christoffersen, Marc Juul; Mai, Quynh-Anh; Tran, Andrew B.; Paull, Morgan; Keasling, Jay D.; Arkin, Adam P.; Endy, Drew
  Nature Methods (2013), 10 (4), 354-360CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
  An inability to reliably predict quant. behaviors for novel combinations of genetic elements limits the rational engineering of biol. systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1000-fold obsd. dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ∼93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain std. biol. parts that support reliable forward engineering of gene expression at genome scales.
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12. 12
  Zhang, X.-Z. and Zhang, Y. H. P. (2011) Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis Microb. Biotechnol. 4, 98– 105
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  12
  Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis
  Zhang Xiao-Zhou; Zhang Y -H Percival
  Microbial biotechnology (2011), 4 (1), 98-105 ISSN:.
  Bacillus subtilis can serve as a powerful platform for directed evolution, especially for secretory enzymes. However, cloning and transformation of a DNA mutant library in B. subtilis are not as easy as they are in Escherichia coli. For direct transformation of B. subtilis, here we developed a new protocol based on supercompetent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids. This new protocol is simple (restriction enzyme-, phosphatase- and ligase-free), fast (i.e. 1 day) and of high efficiency (i.e. ~107 or ~104 transformants per mg of multimeric plasmid or ligated plasmid DNA respectively). Supercompetent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. The DNA mutant library was generated through a two-round PCR: (i) the mutagenized DNA fragments were generated by error-prone PCR and linearized plasmids were made using high-fidelity PCR, and (ii) the multimeric plasmids were generated based on these two DNA templates by using overlap PCR. Both protein expression level and specific activity of glycoside hydrolase family 5 endoglucanse on regenerated amorphous cellulose were improved through this new system. To our limited knowledge, this study is the first report for enhancing secretory cellulase performance on insoluble cellulose.
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13. 13
  Filutowicz, M., McEachern, M. J., and Helinski, D. R. (1986) Positive and negative roles of an initiator protein at an origin of replication Proc. Natl. Acad. Sci. U.S.A. 83, 9645– 9649
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  There is no corresponding record for this reference.
14. 14
  Hiraga, S., Sugiyama, T., and Itoh, T. (1994) Comparative analysis of the replicon regions of eleven ColE2-related plasmids J. Bacteriol. 176, 7233– 7243
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  14
  Comparative analysis of the replicon regions of eleven ColE2-related plasmids
  Hiraga, Shin-Ichiro; Sugiyama, Tomohiko; Itoh, Tateo
  Journal of Bacteriology (1994), 176 (23), 7233-43CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  The incA gene product of ColE2-P9 and ColE3-CA38 plasmids is an antisense RNA that regulates the prodn. of the plasmid-coded Rep protein essential for replication. The Rep protein specifically binds to the origin and synthesizes a unique primer RNA at the origin. The IncB incompatibility is due to competition for the Rep protein among the origins of the same binding specificity. The authors localized the regions sufficient for autonomous replication of 15 ColE plasmids related to ColE2-P9 and ColE3-CA38 (Co1E2-related plasmids), analyzed their incompatibility properties, and detd. the nucleotide sequences of the replicon regions of 9 representative plasmids. The results suggest that all of these plasmids share common mechanisms for initiation of DNA replication and its control. Five IncA specificity types, 4 IncB specificity types, and 9 of the 20 possible combinations of the IncA and IncB types were found. The specificity of interaction of the Rep proteins and the origins might be detd. by insertion or deletion of single nucleotides and substitution of several nucleotides at specific sites in the origins and by apparently corresponding insertion or deletion and substitution of amino acid sequences at specific regions in the C-terminal portions of the Rep proteins. For plasmids of four IncA specificity types, the nine-nucleotide sequences at the loop regions of the stem-loop structures of antisense RNAs are identical, suggesting an evolutionary significance of the sequence. The mosaic structures of the replicon regions with homologous and nonhomologous segments suggest that some of them were generated by exchanging functional parts through homologous recombination.
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15. 15
  Kittleson, J. T., Cheung, S., and Anderson, J. C. (2011) Rapid optimization of gene dosage in E. coli using DIAL strains J. Biol. Eng. 5, 10
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  15
  Rapid optimization of gene dosage in E. coli using DIAL strains
  Kittleson, Joshua T.; Cheung, Sherine; Anderson, J. Christopher
  Journal of Biological Engineering (2011), 5 (), 10CODEN: JBEOBZ; ISSN:1754-1611. (BioMed Central Ltd.)
  Engineers frequently vary design parameters to optimize the behavior of a system. However, synthetic biologists lack the tools to rapidly explore a crit. design parameter, gene expression level, and have no means of systematically varying the dosage of an entire genetic circuit. As a step toward overcoming this shortfall, we have developed a technol. that enables the same plasmid to be maintained at different copy nos. in a set of closely related cells. This provides a rapid method for exploring gene or cassette dosage effects. We engineered two sets of strains to constitutively provide a trans-acting replication factor, either Pi of the R6K plasmid or RepA of the ColE2 plasmid, at different doses. Each DIAL (different allele) strain supports the replication of a corresponding plasmid at a const. level between 1 and 250 copies per cell. The plasmids exhibit cell-to-cell variability comparable to other popular replicons, but with improved stability. Since the origins are orthogonal, both replication factors can be incorporated into the same cell. We demonstrate the utility of these strains by rapidly assessing the optimal expression level of a model biosynthetic pathway for violecein. The DIAL strains can rapidly optimize single gene expression levels, help balance expression of functionally coupled genetic elements, improve investigation of gene and circuit dosage effects, and enable faster development of metabolic pathways.
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16. 16
  Durland, R. H. and Helinski, D. R. (1990) Replication of the broad-host-range plasmid RK2: Direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number J. Bacteriol. 172, 3849– 3858
  [Crossref], [PubMed], [CAS], Google Scholar
  16
  Replication of the broad-host-range plasmid RK2: direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number
  Durland, Ross H.; Helinski, Donald R.
  Journal of Bacteriology (1990), 172 (7), 3849-58CODEN: JOBAAY; ISSN:0021-9193.
  The trfA gene of the broad-host-range plasmid RK2 is essential for initiation of plasmid replication. Two related TrfA proteins of 43 and 32 kDa are produced by independent translation initiation at two start codons within the trfA open reading frame. These proteins were overproduced in Escherichia coli and partially purified. Rabbit antisera raised against the 32-kDa TrfA protein (TrfA-32) and cross-reacting with the 43-kDa protein (TrfA-43) were used in Western blotting (immunoblotting) assays to measure intracellular TrfA levels. In logarithmically growing E. coli HB101, RK2 produced 4.6 ± 0.6 ng of TrfA-32 and 1.8 ± 0.2 ng of TrfA-43 per unit of optical d. at 600 nm (mean ± std. deviation). On the basis of detns. of the no. of cells per unit of optical d. at 600 nm, this corresponds to about 220 mols. of TrfA-32 and 80 mols. of TrfA-43 per cell. Dot blot hybridizations showed that plasmid RK2 is present in about 15 copies per E. coli cell under these conditions. Using plasmid constructs that produce different levels of TrfA proteins, the effect of excess TrfA on RK2 replication was tested. A two- to threefold excess of total TrfA increased the copy no. of RK2 by about 30%. Addnl. increases in TrfA protein concn. had no further effect on copy no., even at levels 170-fold above normal. An RK2 minimal origin plasmid showed a similar response to intracellular TrfA concn. These results demonstrate that TrfA protein concn. is not strictly rate limiting for RK2 replication and that a mechanism that is independent of TrfA concn. functions to limit RK2 copy no. in the presence of excess TrfA.
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17. 17
  Miki, T., Yasukochi, T., Nagatani, H., Furuno, M., Orita, T., Yamada, H., Imoto, T., and Horiuchi, T. (1987) Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: An application to overproduction of chicken lysozyme Protein Eng. 1, 327– 332
  [Crossref], [PubMed], [CAS], Google Scholar
  17
  Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: an application to overproduction of chicken lysozyme
  Miki, Takeyoshi; Yasukochi, Takanori; Nagatani, Hiroko; Furuno, Masahiro; Orita, Tetsuro; Yamada, Hidenori; Imoto, Taiji; Horiuchi, Takao
  Protein Engineering (1987), 1 (4), 327-32CODEN: PRENE9; ISSN:0269-2139.
  A novel expression vector, pKP1500, for synthesizing unfused protein in E. coli was constructed. Plasmid pKP1500 perserves the tac promoter, the lacZ SD sequence, unique restriction sites (EcoRI, SmaI, BamHI, SalI, PstI and HindIII), and the rrnB terminators of pKK223-3, but the replication origin is replaced with that of pUC9. Construction of this plasmid is based upon that the copy no. control of pUC9 is temp. dependent. At 28°, the copy no. of pKP1500 is less than 25 per chromosome, approx. the same copy no. as that of pKK223-3, which contains the replication origin of pBR322, whereas at 42°, the copy no. increases about 10 times and reaches up to 230 copies per chromosome. The main advantage of this system is that the temp.-dependent copy control and regulatable expression of the tac promoter make cells carrying pKP1500 derivs. stable against selective pressure by detrimental overprodn. of foreign proteins at a low temp. and permits high expression of cloned DNAs at a high temp. When chicken lysozyme cDNA carrying the initiation codon (ATG) immediately upstream from the Lys1 codon was inserted downstream from the tac promoter and the SD sequence, the pKP1500 deriv. produced lysozyme at about 25% of the total cellular proteins. This value is more than 10 times higher than that obtained with the pKK223-3 deriv. carrying the same lysozyme cDNA. By comparison, the expression of eukaryotic genes from the tac promoter reported by others has usually been less than a few % of the total cellular protein. Plasmid pKP1500 would, therefore, be useful for the high level prodn. of unfused proteins from eukaryotic cDNAs in E. coli.
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18. 18
  Lin-Chao, S., Chen, W.-T., and Wong, T.-T. (1992) High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II Mol. Microbiol. 6, 3385– 3393
  [Crossref], [PubMed], [CAS], Google Scholar
  18
  High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II
  Lin-Chao, Sue; Chen, Wen Tsuan; Wong, Ten Tsao
  Molecular Microbiology (1992), 6 (22), 3385-93CODEN: MOMIEE; ISSN:0950-382X.
  The plasmids pUC18 and pUC19 are pBR322 derivs. that replicate at a copy no. several fold higher than the parent during growth of Escherichia coli at 37°. The authors show here that the high copy no. of pUC plasmids results from a single point mutation in the replication primer, RNA II, and that the phenotypic effects of this mutation can be suppressed by the Rom (RNA one modulator)/Rop protein or by lowering the growth temp. to 30°. The mutation's effects are enhanced by cell growth at 42°, at which copy no. is further increased. During normal cell growth, the pUC mutation does not affect the length or function of RNA I, the antisense repressor of plasmid DNA replication, but may, as computer anal. suggests, alter the secondary structure of pUC RNA II. The authors suggest that the pUC mutation impedes interactions between the repressor and the primer by producing a temp.-dependent alteration of the RNA II conformation. The Rom/Rop protein may either promote normal folding of the mutated RNA II or, alternatively, may enable the interaction of sub-optimally folded RNA II with the repressor.
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19. 19
  Wong, Q. N. Y., Ng, V. C. W., Lin, M. C. M., Kung, H.-F., Chan, D., and Huang, J.-D. (2005) Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli Nucleic Acids Res. 33, e59
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20. 20
  Acord, J. and Masters, M. (2004) Expression from the Escherichia coli dapA promoter is regulated by intracellular levels of diaminopimelic acid FEMS Microbiol. Lett. 235, 131– 137
  [Crossref], Google Scholar
  There is no corresponding record for this reference.
21. 21
  Datsenko, K. A. and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products Proc. Natl. Acad. Sci. U.S.A. 97, 6640– 6645
  [Crossref], [PubMed], [CAS], Google Scholar
  21
  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
  Datsenko, Kirill A.; Wanner, Barry L.
  Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (12), 6640-6645CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  The authors have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homol. to the targeted gene(s). In this procedure, recombination requires the phage λ Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy no. plasmid. To demonstrate the utility of this approach, the authors generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the resp. PCR products, the authors made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, esp. in genome anal. of E. coli and other bacteria because the procedure can be done in wild-type cells.
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22. 22
  Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K. A., Tomita, M., Wanner, B. L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection Mol. Syst. Biol. 2, 0008
  [Crossref], [PubMed], Google Scholar
  There is no corresponding record for this reference.
23. 23
  Shetty, R. P., Endy, D., and Knight, T. F. (2008) Engineering BioBrick vectors from BioBrick parts J. Biol. Eng. 2, 5
  [Crossref], [PubMed], [CAS], Google Scholar
  23
  Engineering BioBrick vectors from BioBrick parts
  Shetty Reshma P; Endy Drew; Knight Thomas F Jr
  Journal of biological engineering (2008), 2 (), 5 ISSN:.
  BACKGROUND: The underlying goal of synthetic biology is to make the process of engineering biological systems easier. Recent work has focused on defining and developing standard biological parts. The technical standard that has gained the most traction in the synthetic biology community is the BioBrick standard for physical composition of genetic parts. Parts that conform to the BioBrick assembly standard are BioBrick standard biological parts. To date, over 2,000 BioBrick parts have been contributed to, and are available from, the Registry of Standard Biological Parts. RESULTS: Here we extended the same advantages of BioBrick standard biological parts to the plasmid-based vectors that are used to provide and propagate BioBrick parts. We developed a process for engineering BioBrick vectors from BioBrick parts. We designed a new set of BioBrick parts that encode many useful vector functions. We combined the new parts to make a BioBrick base vector that facilitates BioBrick vector construction. We demonstrated the utility of the process by constructing seven new BioBrick vectors. We also successfully used the resulting vectors to assemble and propagate other BioBrick standard biological parts. CONCLUSION: We extended the principles of part reuse and standardization to BioBrick vectors. As a result, myriad new BioBrick vectors can be readily produced from all existing and newly designed BioBrick parts. We invite the synthetic biology community to (1) use the process to make and share new BioBrick vectors; (2) expand the current collection of BioBrick vector parts; and (3) characterize and improve the available collection of BioBrick vector parts.
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24. 24
  Silby, M. W. and Levy, S. B. (2004) Use of in vivo expression technology to identify genes important in growth and survival of Pseudomonas fluorescens Pf0-1 in soil: Discovery of expressed sequences with novel genetic organization J. Bacteriol. 186, 7411– 7419
  [Crossref], [PubMed], [CAS], Google Scholar
  24
  Use of in vivo expression technology to identify genes important in growth and survival of Pseudomonas fluorescens Pf0-1 in soil: Discovery of expressed sequences with novel genetic organization
  Silby, Mark W.; Levy, Stuart B.
  Journal of Bacteriology (2004), 186 (21), 7411-7419CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  Studies were undertaken to det. the genetic needs for the survival of Pseudomonas fluorescens Pf0-1, a gram-neg. soil bacterium potentially important for biocontrol and bioremediation, in soil. In vivo expression technol. (IVET) identified 22 genes with elevated expression in soil relative to lab. media. Soil-induced sequences included genes with probable functions of nutrient acquisition and use, and of gene regulation. Ten sequences, lacking similarity to known genes, overlapped divergent known genes, revealing a novel genetic organization at those soil-induced loci. Mutations in three soil-induced genes led to impaired early growth in soil but had no impact on growth in lab. media. Thus, IVET studies have identified sequences important for soil growth and have revealed a gene organization that was undetected by traditional lab. approaches.
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25. 25
  Varivarn, K., Champa, L. A., Silby, M. W., and Robleto, E. A. (2013) Colonization strategies of Pseudomonas fluorescens Pf0-1: Activation of soil-specific genes important for diverse and specific environments BMC Microbiol. 13, 92
  [Crossref], [PubMed], [CAS], Google Scholar
  25
  Colonization strategies of Pseudomonas fluorescens Pf0-1: activation of soil-specific genes important for diverse and specific environments
  Varivarn, Katila; Champa, Lindsey A.; Silby, Mark W.; Robleto, Eduardo A.
  BMC Microbiology (2013), 13 (), 92CODEN: BMMIBC; ISSN:1471-2180. (BioMed Central Ltd.)
  Background: Pseudomonas fluorescens is a common inhabitant of soil and the rhizosphere environment. In addn. to potential applications in biocontrol and bioremediation, P. fluorescens is of interest as a model for studying bacterial survival and fitness in soil. A previous study using in vivo expression technol. (IVET) identified 22 genes in P. fluorescens Pf0-1 which are up-regulated during growth in Massachusetts loam soil, a subset of which are important for fitness in soil. Despite this and other information on adaptation to soil, downstream applications such as biocontrol or bioremediation in diverse soils remain underdeveloped. We undertook an IVET screen to identify Pf0-1 genes induced during growth in arid Nevada desert soil, to expand our understanding of growth in soil environments, and examine whether Pf0-1 uses general or soil type-specific mechanisms for success in soil environments. Results: Twenty six genes were identified. Consistent with previous studies, these genes cluster in metab., information storage/processing, regulation, and 'hypothetical', but there was no overlap with Pf0-1 genes induced during growth in loam soil. Mutation of both a putative glutamine synthetase gene (Pfl01_2143) and a gene predicted to specify a component of a type VI secretion system (Pfl01_5595) resulted in a decline in arid soil persistence. When examd. in sterile loam soil, mutation of Pfl01_5595 had no discernible impact. In contrast, the Pfl01_2143 mutant was not impaired in persistence in sterile soil, but showed a significant redn. in competitive fitness. Conclusions: These data support the conclusion that numerous genes are specifically important for survival and fitness in natural environments, and will only be identified using in vivo approaches. Furthermore, we suggest that a subset of soil-induced genes is generally important in different soils, while others may contribute to success in specific types of soil. The importance of glutamine synthetase highlights a crit. role for nitrogen metab. in soil fitness. The implication of Type 6 secretion underscores the importance of microbial interactions in natural environments. Understanding the general and soil-specific genes will greatly improve the persistence of designed biocontrol and bioremediation strains within the target environment.
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26. 26
  Molin, S., Boe, L., Jensen, L. B., Kristensen, C. S., Givskov, M., Ramos, J. L., and Bej, A. K. (1993) Suicidal genetic elements and their use in biological containment of bacteria Annu. Rev. Microbiol. 47, 139– 166
  [Crossref], [PubMed], [CAS], Google Scholar
  26
  Suicidal genetic elements and their use in biological containment of bacteria
  Molin, S.; Boe, L.; Jensen, L. B.; Kristensen, C. S.; Givskov, M.; Ramos, J. L.; Bej, A. K.
  Annual Review of Microbiology (1993), 47 (), 139-66CODEN: ARMIAZ; ISSN:0066-4227.
  A review with 70 refs.
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27. 27
  Zielenkiewicz, U. and Ceglowski, P. (2005) The toxin–antitoxin system of the streptococcal plasmid pSM19035 J. Bacteriol. 187, 6094– 6105
  [Crossref], [PubMed], [CAS], Google Scholar
  27
  The toxin-antitoxin system of the streptococcal plasmid pSM19035
  Zielenkiewicz, Urszula; Ceglowski, Piotr
  Journal of Bacteriology (2005), 187 (17), 6094-6105CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  PSM19035 of the pathogenic bacterium Streptococcus pyogenes is a low-copy-no. plasmid carrying erythromycin resistance, stably maintained in a broad range of gram-pos. bacteria. We show here that the ω-ε-ζ operon of this plasmid constitutes a novel protein plasmid addiction system in which the ε and ζ genes encode an antitoxin and toxin, resp., while ω plays an autoregulatory function. Expression of toxin Zeta is bactericidal for the gram-pos. Bacillus subtilis and bacteriostatic for the gram-neg. Escherichia coli. The toxic effects of ζ gene expression in both bacterial species are counteracted by proper expression of ε. The ε-ζ toxin-antitoxin cassette stabilizes plasmids in E. coli less efficiently than in B. subtilis.
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28. 28
  Zielenkiewicz, U., Kowalewska, M., Kaczor, C., and Ceglowski, P. (2009) In vivo interactions between toxin–antitoxin proteins epsilon and zeta of streptococcal plasmid pSM19035 in Saccharomyces cerevisiae J. Bacteriol. 191, 3677– 3684
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  28
  In vivo interactions between toxin-antitoxin proteins Epsilon and Zeta of streptococcal plasmid pSM19035 in Saccharomyces cerevisiae
  Zielenkiewicz, Urszula; Kowalewska, Magdalena; Kaczor, Celina; Ceglowski, Piotr
  Journal of Bacteriology (2009), 191 (11), 3677-3684CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  The widespread prokaryotic toxin-antitoxin (TA) systems involve conditional interaction between two TA proteins. The interaction between the Epsilon and Zeta proteins, constituting the TA system of plasmid pSM19035 from Streptococcus pyogenes, was detected in vivo using a yeast two-hybrid system. As we showed using Saccharomyces cerevisiae, the Zeta toxin hybrid gene also exerts its toxic effects in a dose-dependent manner in eukaryotic cells. Anal. of mutant proteins in the two-hybrid system demonstrated that the N-terminal part of Zeta and the N-terminal region of Epsilon are involved in the interaction. The N-terminal region of the Zeta protein and its ATP/GTP binding motif were found to be responsible for the toxicity.
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29. 29
  de la Cueva-Méndez, G., Mills, A. D., Clay-Farrace, L., Díaz-Orejas, R., and Laskey, R. A. (2003) Regulatable killing of eukaryotic cells by the prokaryotic proteins Kid and Kis EMBO J. 22, 246– 251
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30. 30
  Mutschler, H., Gebhardt, M., Shoeman, R. L., and Meinhart, A. (2011) A novel mechanism of programmed cell death in bacteria by toxin–antitoxin systems corrupts peptidoglycan synthesis PLoS Biol. 9, e1001033
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31. 31
  Pimentel, B., Madine, M. A., and de la Cueva-Méndez, G. (2005) Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1 EMBO J. 24, 3459– 3469
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32. 32
  Salis, H. M., Mirsky, E. A., and Voigt, C. A. (2009) Automated design of synthetic ribosome binding sites to control protein expression Nat. Biotechnol. 27, 946– 950
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  32
  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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33. 33
  Casini, A., MacDonald, J. T., Jonghe, J. D., Christodoulou, G., Freemont, P. S., Baldwin, G. S., and Ellis, T. (2013) One-pot DNA construction for synthetic biology: The Modular Overlap-Directed Assembly with Linkers (MODAL) strategy Nucleic Acids Res. 42, e7
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34. 34
  de la Cueva-Méndez, G. and Pimentel, B. (2007) Gene and cell survival: Lessons from prokaryotic plasmid R1 EMBO Rep. 8, 458– 464
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35. 35
  Olson, D. G. and Lynd, L. R. (2012) Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles J. Biol. Eng. 6, 5
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  Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles
  Olson, Daniel G.; Lynd, Lee R.
  Journal of Biological Engineering (2012), 6 (), 5CODEN: JBEOBZ; ISSN:1754-1611. (BioMed Central Ltd.)
  Background: Temp.-sensitive (Ts) plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram pos., thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. for predicting Ts mutants based on the amino-acid sequence of the protein. Results: A library of 34 mutant plasmids was designed, synthesized and screened, resulting in 6 mutants which exhibited a Ts phenotype. Of these 6, the one with the most temp.-sensitive phenotype (M166A) was compared with the original plasmid. It exhibited lower stability at 48°C and was completely unable to replicate at 55°C. Conclusions: The plasmid described in this work could be useful in future efforts to genetically engineer C. thermocellum and the method used to generate this plasmid may be useful for others trying to make Ts plasmids.
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36. 36
  Pellegrini, O., Mathy, N., Gogos, A., Shapiro, L., and Condon, C. (2005) The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor Mol. Microbiol. 56, 1139– 1148
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  The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor
  Pellegrini, Olivier; Mathy, Nathalie; Gogos, Arhonda; Shapiro, Lawrence; Condon, Ciaran
  Molecular Microbiology (2005), 56 (5), 1139-1148CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
  Operons encoding stable toxins and their labile antidote are widespread in prokaryotes and play important roles in plasmid partitioning and cellular responses to stress. One such family of toxins MazF/ChpAK/PemK encodes an endoribonuclease that inactivates cellular mRNAs by cleaving them at specific, but frequently occurring sites. Here the authors show that the Bacillus subtilis ydcE gene encodes a member of this family of RNases, which the authors have called EndoA. Overexpression of EndoA is toxic for bacterial cell growth and this toxicity is reversed by coexpression of the gene immediately upstream, ydcD. Furthermore, YdcD inhibits EndoA activity directly in vitro. EndoA has similar cleavage specificity to MazF and PemK and yields cleavage products with 3'-phosphate and 5'-hydroxyl groups, typical of EDTA-resistant degradative RNases. This is the first example of an antitoxin-toxin system in B. subtilis.
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37. 37
  Wang, B., Barahona, M., and Buck, M. (2013) A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals Biosens. Bioelectron. 40, 368– 376
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38. 38
  Friedland, A. E., Lu, T. K., Wang, X., Shi, D., Church, G., and Collins, J. J. (2009) Synthetic gene networks that count Science 324, 1199– 1202
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  Synthetic Gene Networks That Count
  Friedland, Ari E.; Lu, Timothy K.; Wang, Xiao; Shi, David; Church, George; Collins, James J.
  Science (Washington, DC, United States) (2009), 324 (5931), 1199-1202CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one the ability to program and design cells with some of the principles of modern computing, such as counting. A cellular counter would enable complex synthetic programming and a variety of biotechnol. applications. Here, we report two complementary synthetic genetic counters in Escherichia coli that can count up to three induction events: the first, a riboregulated transcriptional cascade, and the second, a recombinase-based cascade of memory units. These modular devices permit counting of varied user-defined inputs over a range of frequencies and can be expanded to count higher nos.
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39. 39
  Callura, J. M., Dwyer, D. J., Isaacs, F. J., Cantor, C. R., and Collins, J. J. (2010) Tracking, tuning, and terminating microbial physiology using synthetic riboregulators Proc. Natl. Acad. Sci. U.S.A. 107, 15898– 15903
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40. 40
  Ronchel, M. C. and Ramos, J. L. (2001) Dual system to reinforce biological containment of recombinant bacteria designed for rhizoremediation Appl. Environ. Microbiol. 67, 2649– 2656
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41. 41
  Neuhard, J., Price, A. R., Schack, L., and Thomassen, E. (1978) Two thymidylate synthetases in Bacillus subtilis Proc. Natl. Acad. Sci. U.S.A. 75, 1194– 1198
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42. 42
  Marris, C. and Jefferson, C. (2013) Synthetic Biology: Containment and Release of Engineered Micro-organisms. Workshop held at King’s College London, 29th April 2013. Summary of discussions available from http://www.kcl.ac.uk/sspp/departments/sshm/research/Research-Labs/CSynBi-Events.aspx.
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43. 43
  World Health Organization. ( (2011) Guidelines for Drinking-Water Quality, 4th ed., WHO Press, Geneva. Available from http://www.who.int/.
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44. 44
  Kang, H. Y., Dozois, C. M., Tinge, S. A., Lee, T. H., and Curtiss, R., III (2002) Transduction-mediated transfer of unmarked deletion and point mutations through use of counterselectable suicide vectors J. Bacteriol. 184, 307– 312
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  Transduction-mediated transfer of unmarked deletion and point mutations through use of counterselectable suicide vectors
  Kang, Ho Young; Dozois, Charles M.; Tinge, Steven A.; Lee, Tae Ho; Curtiss, Roy, III
  Journal of Bacteriology (2002), 184 (1), 307-312CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  A challenge in strain construction is that unmarked deletion and nucleotide substitution alleles generally do not confer selectable phenotypes. We describe here a rapid and efficient strategy for transferring such alleles via generalized transduction. The desired allele is first constructed and introduced into the chromosome by conventional allelic-exchange methods. The suicide vector contg. the same allele is then integrated into the mutant chromosome, generating a tandem duplication homozygous for that allele. The resulting strain is used as a donor for transductional crosses, and selection is made for a marker carried by the integrated suicide vector. Segregation of the tandem duplication results in haploid individuals, each of which carries the desired allele. To demonstrate this mutagenesis strategy, we used bacteriophage P22HTint for generalized transduction-mediated introduction of unmarked mutations to Salmonella enterica serovar Typhimurium. This method is applicable to any species for which generalized transduction is established.
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45. 45
  Sat, B., Reches, M., and Engelberg-Kulka, H. (2003) The Escherichia coli mazEF suicide module mediates thymineless death J. Bacteriol. 185, 1803– 1807
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  The Escherichia coli mazEF suicide module mediates thymineless death
  Sat, Boaz; Reches, Myriam; Engelberg-Kulka, Hanna
  Journal of Bacteriology (2003), 185 (6), 1803-1807CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  In 1954, Cohen and Barner discovered that a thymine auxotrophic (thyA) mutant of Escherichia coli undergoes cell death in response to thymine starvation. This phenomenon, called thymineless death (TLD), has also been found in many other organisms, including prokaryotes and eukaryotes. Though TLD has been studied intensively, its mol. mechanism has not yet been explained. Previously we reported on the E. coli mazEF system, a regulatable chromosomal suicide module that can be triggered by various stress conditions. MazF is a stable toxin, and MazE is an unstable antitoxin. Here, we show that cell death that is mediated by the mazEF module can also be activated by thymine starvation. We found that TLD depends on E. coli mazEF and that under thymine starvation, the activity of the mazEF promoter P2 is significantly reduced. Our results, which describe thymine starvation as a trigger for a built-in death program, have implications for programmed cell death in both prokaryotes and eukaryotes.
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46. 46
  Steidler, L., Neirynck, S., Huyghebaert, N., Snoeck, V., Vermeire, A., Goddeeris, B., Cox, E., Remon, J. P., and Remaut, E. (2003) Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10 Nat. Biotechnol. 21, 785– 789
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  46
  Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10
  Steidler, Lothar; Neirynck, Sabine; Huyghebaert, Nathalie; Snoeck, Veerle; Vermeire, An; Goddeeris, Bruno; Cox, Eric; Remon, Jean Paul; Remaut, Erik
  Nature Biotechnology (2003), 21 (7), 785-789CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
  Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease. However, the release of such genetically modified organisms through clin. use raises safety concerns. In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene. This thyA- hIL10+ L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biol. containment system and the bacterium's capacity to secrete hIL-10 were validated in vivo in pigs. Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strain acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.
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47. 47
  Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., and Mattick, J. S. (1991) Touchdown PCR to circumvent spurious priming during gene amplification Nucleic Acids Res. 19, 4008
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48. 48
  Stemmer, W. P., Crameri, A., Ha, K. D., Brennan, T. M., and Heyneker, H. L. (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides Gene 164, 49– 53
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  Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides
  Stemmer, Willem P. C.; Crameri, Andreas; Ha, Kim D.; Brennan, Thomas M.; Heyneker, Herbert L.
  Gene (1995), 164 (1), 49-53CODEN: GENED6; ISSN:0378-1119. (Elsevier)
  Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large nos. of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling (Stemmer, W.P.C. 1994), does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment contg. the TEM-1 β-lactamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene was PCR amplified and cloned in a vector contg. the tetracycline-resistance gene (TcR) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the TcR colonies were ApR, making this approach a general method for the rapid and cost-effective synthesis of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel contg. 134 oligos, a high-mol.-mass multimeric form of a 2.7-kb plasmid contg. the bla gene, the α-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.
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49. 49
  Kuhlman, T. E. and Cox, E. C. (2010) Site-specific chromosomal integration of large synthetic constructs Nucleic Acids Res. 38, e92
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  Site-specific chromosomal integration of large synthetic constructs
  Kuhlman, Thomas E.; Cox, Edward C.
  Nucleic Acids Research (2010), 38 (6), e92/1-e92/10CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
  The authors have developed an effective, easy-to-use two-step system for the site-directed insertion of large genetic constructs into arbitrary positions in the Escherichia coli chromosome. The system uses λ-Red mediated recombineering accompanied by the introduction of double-strand DNA breaks in the chromosome and a donor plasmid bearing the desired insertion fragment. This method, in contrast to existing recombineering or phage-derived insertion methods, allows for the insertion of very large fragments into any desired location and in any orientation. This method was demonstrated by inserting a 7-kb fragment consisting of a venus-tagged lac repressor gene along with a target lacZ reporter into six unique sites distributed sym. about the chromosome. The universality and repeatability of the method was shown by sep. inserting the lac repressor gene and the lacZ target into the chromosome at sep. locations around the chromosome via repeated application of the protocol.
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50. 50
  Imamura, N. and Nakayama, H. (1982) thiK and thiL loci of Escherichia coli J. Bacteriol. 151, 708– 717
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  thiK and thiL loci of Escherichia coli
  Imamura, Nariko; Nakayama, Hideo
  Journal of Bacteriology (1982), 151 (2), 708-17CODEN: JOBAAY; ISSN:0021-9193.
  Mutants of E. coli K-12 auxotrophic for thiamin phosphates were produced in stepwise fashion from the polyauxotrophic F- strain JC1552, via intermediate prodn. of thiamin auxotrophs that had lost the enzymic activity of either phosphomethylpyrimidine kinase or thiamin phosphate pyrophosphorylase. They include 2 types: one responds to thiamin monophosphate or thiamin pyrophosphate, and the other responds to thiamin pyrophosphate only; the former lacks thiamin kinase activity, and the latter lacks thiamin monophosphate kinase activity, in addn. to the enzymic defects caused by the 1st mutations. Two new genes were found for which the designations thiK and thiL are proposed, which govern the activities of thiamin kinase and thiamin monophosphate kinase, resp. By conjugation and P1 transduction, the thiK locus was mapped at ∼25 min, between pyrC and purB and close to fabD. The relative order of thiK with respect to nearby genes was tentatively established as pyrC-pstG-fabD-thiK-purB. In the case of thiL, the locus was situated at ∼9 min, between tsx and acrA and probably 0.2 min clockwise from the former.
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51. 51
  Webb, E. and Downs, D. (1997) Characterization of thiL, encoding thiamin-monophosphate kinase Salmonella typhimurium J. Biol. Chem. 272, 15702– 15707
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52. 52
  Schyns, G., Potot, S., Geng, Y., Barbosa, T. M., Henriques, A., and Perkins, J. B. (2005) Isolation and characterization of new thiamine-deregulated mutants of Bacillus subtilis J. Bacteriol. 187, 8127– 8136
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  Isolation and characterization of new thiamine-deregulated mutants of Bacillus subtilis
  Schyns, Ghislain; Potot, Sebastien; Geng, Yi; Barbosa, Teresa M.; Henriques, Adriano; Perkins, John B.
  Journal of Bacteriology (2005), 187 (23), 8127-8136CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  In bacteria, thiamin pyrophosphate (TPP) is an essential cofactor that is synthesized de novo. Thiamin, however, is not an intermediate in the biosynthetic pathway but is salvaged from the environment and phosphorylated to TPP. We have isolated and characterized new mutants of Bacillus subtilis that deregulate thiamin biosynthesis and affect the export of thiamin products from the cell. Deletion of the ydiA gene, which shows significant similarity to the thiamin monophosphate kinase gene of Escherichia coli (thiL), did not generate the expected thiamin auxotroph but instead generated a thiamin bradytroph that grew to near-wild-type levels on minimal medium. From this ΔthiL deletion mutant, two addnl. Et methanesulfonate-induced mutants that derepressed the expression of a thiC-lacZ transcriptional reporter were isolated. One mutant, Tx1, contained a nonsense mutation within the B. subtilis yloS (thiN) gene that encodes a thiamin pyrophosphokinase, a result which confirmed that B. subtilis contains a single-step, yeast-like thiamin-to-TPP pathway in addn. to the bacterial TPP de novo pathway. A second mutant, strain Tx26, was shown to contain two lesions. Genetic mapping and DNA sequencing indicated that the first mutation affected yuaJ, which encodes a thiamin permease. The second mutation was located within the ykoD cistron of the ykoFEDC operon, which putatively encodes the ATPase component of a unique thiamin-related ABC transporter. Genetic and microarray studies indicated that both the mutant yuaJ and ykoD genes were required for the derepression of thiamin-regulated genes. Moreover, the combination of the four mutations (the ΔthiL, thiN, yuaJ, and ykoD mutations) into a single strain significantly increased the prodn. and excretion of thiamin products into the culture medium. These results are consistent with the proposed "riboswitch" mechanism of thiamin gene regulation.
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53. 53
  Domínguez, M. C., La Rosa, de M., and Borobio, M. V. (2001) Application of a spectrophotometric method for the determination of post-antibiotic effect and comparison with viable counts in agar J. Antimicrob. Chemother. 47, 391– 398
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  Application of a spectrophotometric method for the determination of post-antibiotic effect and comparison with viable counts in agar
  Dominguez, M. Carmen; De la Rosa, Manuel; Borobio, M. Victoria
  Journal of Antimicrobial Chemotherapy (2001), 47 (4), 391-398CODEN: JACHDX; ISSN:0305-7453. (Oxford University Press)
  The post-antibiotic effects of gentamicin and ciprofloxacin at 1 ×, 2 × and 4 × MIC on Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 were studied using a spectrophotometric method and the classic method of viable counts on agar as a ref. Monitoring of the growth kinetics was carried out by viability counting on the plate every hour and by means of the optical d. of the cultures measured by spectrophotometry at a wavelength of 450 nm. No statistically significant differences were found between the results obtained with the spectrophotometric method and the ref. method. The former method was much quicker, much easier to use and to replicate.
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54. 54
  Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680– 685
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  54
  Cleavage of structural proteins during the assembly of the head of bacteriophage T4
  Laemmli, U. K.
  Nature (London, United Kingdom) (1970), 227 (5259), 680-685CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
  Using an improved method of polyacrylamide gel electrophoresis based on the capability of SDS to break down proteins into their individual polypeptide chains, many previously unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
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55. 55
  Bravo, A., Torrontegui, G., and Díaz, R. (1987) Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid Mol. Gen. Genet. 210, 101– 110
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  55
  Identification of components of a new stability system of plasmid R1, ParD, that is close to to the origin of replication of this plasmid
  Bravo, Alicia; De Torrontegui, Gertrudis; Diaz, Ramon
  Molecular and General Genetics (1987), 210 (1), 101-10CODEN: MGGEAE; ISSN:0026-8925.
  A mutation which derepresses an autoregulated system that is located in the vicinity of the basic replicon of R1 stabilizes the ParA- and ParB- miniplasmid of R1, pKN1562, without increasing its copy no. The system, called ParD, maps inside the 1.45-kb PstI-EcoRI fragment that is adjacent to the origin of replication of the plasmid. Two proteins whose expression is coordinated are components of the system. The sequence of the PstI-EcoRI fragment was obtained. The wild-type ParD system dets. in cis a basal but detectable stability.
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Supporting Information
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Supplementary figures, sequence tables, GenBank accession numbers, and annotated plasmid ApE files (ApE can be downloaded for free at http://biologylabs.utah.edu/jorgensen/wayned/ape/). This material is available free of charge via the Internet at http://pubs.acs.org. In addition, selected GeneGuard plasmids may be obtained from the SEVA repository by request (see http://seva.cnb.csic.es/).
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GeneGuard: A Modular Plasmid System Designed for Biosafety

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Abstract

Results and Discussion

Conditional Origins of Replication (CORs)

Figure 1

Rich-media Compatible Auxotrophs

Figure 2

Toxin–Antitoxin Systems As a Negative Selection Pressure

Figure 3

GeneGuard Genomic and Vector Cassettes

Figure 4

Using the GeneGuard System for Heavy-Metal Biosensors

Figure 5

Methods

Media and General Materials

Device Construction

Flow Cytometry

Genomic Integration

Plasmid Stability Assay

Soil Assay

Growth Curves

Western Blotting

Transformation Assay

Biosensor Assay

Supporting Information

Terms & Conditions

Author Information

Acknowledgment

References

Cited By

Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

References

Supporting Information

Supporting Information

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