Overexpression of the tcp Gene Cluster Using the T7 RNA Polymerase/Promoter System and Natural Transformation-Mediated Genetic Engineering of Vibrio cholerae

The human pathogen and aquatic bacterium Vibrio cholerae belongs to the group of naturally competent bacteria. This developmental program allows the bacterium to take up free DNA from its surrounding followed by a homologous recombination event, which allows integration of the transforming DNA into the chromosome. Taking advantage of this phenomenon we genetically engineered V. cholerae using natural transformation and FLP recombination. More precisely, we adapted the T7 RNA polymerase/promoter system in this organism allowing expression of genes in a T7 RNA polymerase-dependent manner. We naturally transformed V. cholerae by adding a T7-specific promoter sequence upstream the toxin-coregulated pilus (tcp) gene cluster. In a V. cholerae strain, which concomitantly produced the T7 RNA polymerase, this genetic manipulation resulted in the overexpression of downstream genes. The phenotypes of the strain were also in line with the successful production of TCP pili. This provides a proof-of-principle that the T7 RNA polymerase/promoter system is functional in V. cholerae and that genetic engineering of this organism by natural transformation is a straightforward and efficient approach.


Introduction
A plethora of methods exist today to allow genetic engineering of bacteria. Many of those methods have been used for the last 30 years or even longer and straightforward protocols are available today to master those techniques. One prominent example is the seminal book Molecular Cloning: A Laboratory Manual published initially in 1982 by Tom Maniatis, Edward Fritsch, and Joseph Sambrook [1] (the currently available 4 th edition is authored by Michael R. Green and Joseph Sambrook; http:// www.molecularcloning.com). But even though many techniques are available and work well, reducing the time needed for cloning is often desired. Thus, we developed a fast protocol [2][3][4] to genetically modify our favorite organism, the human pathogen and aquatic bacterium Vibrio cholerae, using chitin-induced natural transformation [5,6].
The regulatory network of chitin-induced natural transformation of V. cholerae is extremely complicated and brings together the pathways of chitin sensing and degradation, quorum sensing, and carbon catabolite repression (summarized in [7,8] and recently reviewed in [9]). However, the advantage of the DNA uptake process of V. cholerae over some other naturally competent Gramnegative bacteria such as Haemophilus influenzae and Neisseria gonorrhoeae is that V. cholerae is not fastidious about the kind of DNA it takes up while it is competent [10]. This makes natural competence and transformation a perfect system for fast and efficient delivery of DNA to the cells. In this study we applied our previously published protocol describing the combination of natural transformation and FLP recombination in V. cholerae (TransFLP, [3,4]) in order to integrate DNA sequences in a sitespecific manner onto the chromosome. The rational behind this study was to create V. cholerae strains in which we were able to artificially express large gene clusters. We reasoned that it would be difficult or even impossible to clone such gene clusters onto plasmids/cosmids thereby expressing the genes in trans. Furthermore, as many of the common plasmids seem to be quickly lost from V. cholerae without constant selection pressure plasmidencoded expression was excluded from this study. Consequently, we decided to express our gene cluster of interest by adding a strong and specific promoter in cis of the indigenous genes on the chromosome using our TransFLP method (Fig. 1).
To our knowledge the strongest and most exclusive expression system described so far in bacteria is based on the T7 RNA polymerase/promoter combination. Several studies in the 1980's have led to the establishment of this very successful system [11,12], which is still used our days (mostly with variations from the original protocols). The gene encoding the T7 DNA-directed RNA polymerase (EC 2.7.7.6) (from here on referred to as T7 RNA polymerase), phage T7 gene 1, was first cloned by Davanloo et al. in 1984 [13]. This opened up new possibilities as the T7 RNA polymerase ''has a stringent specificity for its own promoters and will selectively transcribe DNA that has been linked to such a promoter'' [13]. Such specificity was considered useful for directing the expression of only a subset of genes within a cell. The T7 RNA polymerase-dependent promoter is a ''highly conserved sequence of 23 continuous base pairs including the start site for the RNA'' [13]. Few if any of these conserved sequences are found in bacterial hosts causing this stringent specificity of the enzyme. The 22-23 base pairs consensus sequence of the T7 RNA polymerase promoter has been previously studied [14][15][16]. However, Ikeda et al. identified the essential bases of this specific promoter sequence by screening a randomly mutagenized library of T7 promoter sequences and correlating the in vivo activity to the sequences [17]. In our study the T7 W10 promoter was used, which encompasses 28 bp: GAAATTAATACGACTCACTA-TAGGGAGA [17,18]. We choose this strong expression system to have the best possible outcome as our study is aimed at proof of principle. Below we explain the cloning strategy and the experimental execution in detail. Furthermore, we provide evidence that the target gene cluster was indeed highly expressed in the genetically engineered strain, which resulted in the desired phenotypes. Obstacles and future needs for improvement are also discussed.
The target of our first trial was the toxin-coregulated pilus (tcp) [19] gene cluster of V. cholerae. The tcp gene cluster is located on the Vibrio pathogenicity island 1 (VPI-1) [20] previously termed TCP-ACF element [21]. The reason for this choice was that this cluster has been extensively studied in vivo and also under virulence inducing conditions in vitro (e.g. through the use of AKI growth conditions [22,23]). The TCP pilus is essential for intestinal colonization as it allows the establishment of microcolonies in the host [19], which was shown both in humans and in the infant mouse animal model of cholera [24][25][26][27]. The bundle-forming TCP pilus is a member of Type IVb pili, which ''are found almost exclusively on enteric bacteria'' [28]. The TCP pilus (classical and El Tor type) has been characterized based on mutant analysis, electron microscopy (EM; such as TEM, SEM, STEM, and threedimensional high-resolution field emission scanning electron microscopy, FESEM), crystallization of the major pilin TcpA, and CryoEM reconstruction of full length TCP [28][29][30][31][32]. Based on in vivo and in vitro data it was concluded that a major function of this pilus is to mediate interactions between bacteria through microcolony formation that is required for intestinal colonization [30]. Indeed, Kirn et al. provided evidence that autoagglutination correlates well with intestinal colonization in the infant mouse model [30]. Thus, we hypothesized that induced expression of the tcp cluster would provide us with phenotypes such as in vitro autoagglutination/microcolony formation due to TCP production. It should be noted that artifical induction of the tcp cluster was already performed earlier by expressing toxT, which encodes the major transcriptional activator of virulence genes in V. cholerae [33], from an inducible promoter [29]. This method led to the production of the major subunit of TCP, TcpA, both in the classical and El Tor biotypes of V. cholerae. Furthermore, TCP fibers of V. cholerae O1 biotype El Tor could be visualized by EM upon toxT overexpression [29]. However, whereas autoagglutination of the classical biotype strain was readily observable under such artificial induction conditions, autoagglutination of a V. cholerae O1 El Tor strain (C6706) did not occur [31]. The author of this study speculated that the reduced level of TcpA protein and TCP fibers (,50%) for the O1 El Tor biotype compared to the classical strain O395 could partially explain this lack of autoagglutination [31]. To circumvent this issue, we aimed at high-level expression of the whole tcp cluster by directly inducing this cluster using the T7 RNA polymerase dependent system described above. The promoter sequence was thereby introduced upstream of the tcpA gene in a site-directed manner using the natural transformation-based TransFlp method as a tool [3,4]. Proof-of-principle was provided by successful TCP-mediated agglutination, microcolony formation, and the visualization of TCP fibers using scanning electron microscopy (SEM). The rational behind this study was to engineer V. cholerae strains so that large gene clusters can be artificially expressed independent of growth condition restraints. The idea was to integrate the lacUV5-promoter controlled T7 RNA polymerase-encoded gene together with its repressor gene (lacI; both derived from E. coli BL21(DE3) [12]) into the V. cholerae chromosome. Using this strain, specific genes could be put under control of the T7 RNA polymerase solely by integrating the T7 RNA polymerase-dependent promoter sequence (indicated in black box; P T7 ) at the respective locus on the chromosome using natural transformation. Alternatively, the P T7 sequence was integrated on a plasmid as is commonly done in E. coli overexpression systems. doi:10.1371/journal.pone.0053952.g001

Materials and Methods
Bacterial strains and plasmids V. cholerae strains and plasmids used in this study are indicated in Table 1. E. coli strains DH5a [34] and BL21(DE3) [12] were used as hosts for cloning purposes and to test T7 RNA polymerasedependent expression constructs. Bacterial mating between V.
cholerae and E. coli was done using E. coli strain S17-1lpir [35] as the donor strain.

Media and growth conditions
Overnight cultures of V. cholerae and E. coli strains were grown in LB medium with shaking at 30uC. Bacterial strains used for gene expression profiling, phenotypic characterization and SEM were also cultured in LB either in the absence or in the presence of  1 mM IPTG. Ampicillin was added for plasmid maintenance at a concentration of 100 mg/ml whenever required. Fifty mg/ml gentamicin was used to select mTn7-T7RNAP-containing V. cholerae cells after bacterial mating with E. coli cells. E. coli cells were counter-selected by using Vibrio selective medium (TCBS; Fluka).

Construction of mini-Tn7 transposon carrying the gene encoding for T7 RNA polymerase
Plasmid pGP704-mTn7-T7RNAP was generated by ligating the SmaI-digested vector pGP704::Tn7 (Table 1) with the lacI-P lacUV -T7RNAP cluster-containing and ScaI-digested PCR fragment. PCR was performed using primer pair LacI-before and T7 RNA pol after (Table S1) and genomic DNA of E. coli strain BL21(DE3) as template [12]. The construct was verified by sequencing.

Construction of T7 RNA polymerase-dependent transcriptional reporter plasmid
Plasmid pBR-[P T7 ]-GFP was constructed as follows. The gfp coding region (including the Shine-Dalgarno sequence) was PCRamplified using oligonucleotides P[T7]-GFP-up-P and P[T7]-GFP-down-BamHI (Table S1) and genomic DNA of strain A1552-GFP [36] as template. The upstream primer had a 59 phosphorylated and non-priming overhang containing the T7 RNA polymerase dependent promoter sequence (as indicated in Fig. 1). The fragment was digested with BamHI and cloned into EcoRV/ BamHI double-digested vector pBR-Tet_MCSI [7]. Correct ligation was validated by colony PCR and sequencing.
The T7 RNA polymerase-dependent promoter sequence was site-specifically integrated into the chromosome using natural transformation and FLP recombination (TransFLP method; [3,4]).
Oligonucleotides for the design of the transforming PCR fragment are indicated in Table S1. The ctx operon was likewise deleted (using PCR fragment DctxAB-FRT-Kan-FRT; primer indicated in Table S1).

Quantitative reverse transcription PCR (qRT-PCR)
V. cholerae strains were grown for several hours in LB containing 1mM IPTG until they reached an optical density at 600 nm of ,2. IPTG-induced strain ADctxAB-[P T7 ]-tcp-T7RNAP was harvested at the same time though the OD 600 values varied due to extensive clumping of the cells (autoagglutination as described in the result section). Harvesting of cells, RNA preparation, reverse transcription and qPCR were performed as previously described [7]. Expression levels were normalized to the housekeeping gene gyrA. Primers used for qRT-PCR are listed in Table S1.

Epifluorescence microscopy
Visualization of bacterial cells at the microscopic level was done using a Zeiss Axio Imager M2 epifluorescence microscope. Image acquisition was performed using the Zeiss AxioVision software steering a high-resolution camera (AxioCam MRm). Images were rotated, cropped and magnified using Zeiss AxioVision and ImageJ.

Scanning electron microscopy (SEM)
Bacterial cells were grown as described. At the time of harvest, cells were spotted onto a silicon wafer. Attachment was allowed to occur for several minutes before the cells were fixed for 1 hour with 1.25% glutaraldehyde and 1% tannic acid in 0.1 M phosphate buffer, pH 7.4. Cells were washed in phosphate buffered saline and then further fixed for 30 minutes in 1.0% osmium tetroxide in the same buffer. The bacteria, attached to the wafers, were dehydrated in a graded alcohol series and then dried by passing through the supercritical point of carbon dioxide (Leica Microsystems CPD300). The samples were coated with a 2 nm layer of osmium using an osmium plasma coater (Filgen OPC60). Images of the bacteria were taken with a field emission scanning electron microscope (Merlin, Zeiss NTS) using an acceleration voltage of 2 kV and the in-lens secondary electron detector.

Results and Discussion
The aim of this study was to genetically engineer V. cholerae strains so that large gene clusters could be artificially expressed in vitro. Consequently, the experimental design was as follows (Fig. 1): First, we integrated the T7 RNA polymerase encoding gene (T7 gene 1) preceded by the lacUV5 promoter and the Lac repressor gene (lacI) into the V. cholerae chromosome. The T7 specific promoter sequence was delivered on a plasmid (control) as well as upstream the tcp operon. The latter was accomplished using chitininduced natural transformation followed by FLP recombination. Expression of tcp and expected phenotypes were confirmed.
Integration of T7 RNA polymerase encoding gene into the V. cholerae chromosome As the T7 RNA polymerase has a very stringent specificity towards the T7-specific promoter sequences [40] we decided to integrate the encoding gene (T7 gene 1; [13]) into the V. cholerae genome (Fig. 1). To do so we amplified the T7 gene 1 preceded by the lacUV5 promoter and the lac repressor gene lacI from the l(DE3) prophage contained in the lysogenic E. coli strain BL21(DE3) [12] using PCR and primers LacI-before and T7 RNA pol after (Table S1). After restriction enzyme digestion (see Material and Methods) the PCR fragment was ligated into the likewise digested miniTn7 transposon [37] harbored on plasmid pGP704::Tn7 [41]. After triparental mating between the E. coli strains S17-1lpir/pGP704-mTn7-T7RNAP (carrying the construct-containing transposon), S17-1lpir/pUX-BF13 (providing the Tn7 transposition in trans [37]), and V. cholerae wild type strain  [46] and based on [47]; not to scale). Middle row: the transforming PCR-derived DNA fragment included parts of the genes tcpH and tcpA as flanking regions (in blue) to allow homologous recombination with the chromosome. In addition the PCR fragment carried the FRT-site (red rectangles) flanked kanamycin resistant cassette (aph; green arrow) and the T7 RNA polymerase-dependent promoter sequence (black box; according to Fig. 1). Lower row: the structure of the VPI-1 island after natural transformation and FLP recombination of the WT strain using the PCR fragment indicated in the middle row as transforming DNA material. B: PCRbased verification of site-directed insertion. The T7 RNA polymerase-dependent promoter sequence was inserted into the V. cholerae genome by chitin-induced natural transformation followed by FLP-mediated excision of the antibiotics resistance cassette (TransFLP; [3,4]). The correctness of the resulting strain was tested by PCR using primer pair

Functionality of T7 RNA polymerase in V. cholerae
In order to test whether the T7 RNA polymerase gene was functional in V. cholerae a transcriptional reporter gene was cloned onto a plasmid (Table 1). To accomplish this, the green florescent protein-encoding gene (gfp) was PCR-amplified from V. cholerae strain A1552-GFP [36]. Concomitantly with this amplification step we added the T7 promoter sequence ''GAAATTAATAC-GACTCACTATAGGGAGA'' (Fig. 1) upstream the gfp coding region by incorporating this sequence as 59-overhang into the forward primer (Table S1). The respective P T7 -gfp fragment was ligated in the promoterless plasmid pBR-Tet_MCSI [7] yielding plasmid pBR-[P T7 ]-gfp ( Table 1). The functionality of this plasmid was first tested in the well-characterized E. coli strain BL21(DE3) [12] (Fig. 2) as this strain has been extensively used over the years for T7 RNA polymerase dependent high-level expression of proteins. The respective E. coli transformant was grown in rich medium either in the absence or presence of the inducer IPTG and T7 RNA polymerase-dependent expression was visualized using epifluorescence microscopy (Fig. 2). Though low-level expression of gfp was also observed under uninduced growth conditions (as discussed below), the expression was significantly enhanced upon provision of the inducer. Thus, the functionality and usefulness of the reporter plasmid was established.
Next, the reporter plasmid was transferred in two different V. cholerae strains: the wild type strain A1552 and the newly constructed strains AT7RNAP (T7RNAP + ) (Fig. 3). Similar to the experiment described above for E. coli, we grew both transformants in rich medium without or with IPTG as the inducer of T7 RNA polymerase production. Whereas no green fluorescent signal was detectable under both conditions in the V. cholerae strain lacking T7 RNA polymerase (Fig. 3, left), gfp expression was unambiguously detectable in strain AT7RNAP and significantly enhanced in the presence of the inducer IPTG (Fig. 3,  right). We conclude that the T7 RNA polymerase was functional in V. cholerae and that T7 RNA polymerase specific promoter sequences were not unspecifically transcribed (or at least not at high levels) in V. cholerae strains lacking T7 RNA polymerase. Thus, the system seemed feasible for induction of large gene clusters in a T7 RNA polymerase-dependent manner.
Integration of the T7 RNA polymerase specific promoter sequence upstream tcpA using the TransFLP method As described above and as a proof-of-principle we were interested in artificially expressing the tcp cluster in vitro under non-virulence inducing conditions. As it has previously been shown that induced expression of toxT led to enhanced TcpA and cholera toxin (CT) production [29] and as toxT is embedded within the tcp cluster (Fig. 4) we decided to first attenuate the newly constructed V. cholerae strain (AT7RNAP) by deleting the ctxAB operon using chitin-induced TransFLP (data not shown) [2][3][4]. The resulting strain was named ADctxAB-T7RNAP (Table 1). Next, to create an artificial induction system for the tcp gene cluster we needed to integrate the T7 RNA polymerase specific promoter sequence (Fig. 1) upstream the first gene whose gene product is directly involved in the toxin-coregulated pilus biosynthesis, namely tcpA (Fig. 4A). We did so by creating a PCR fragment consisting of the following features (Fig. 4A, middle row): 1) an FRT-site flanked antibiotic resistance cassette (here aph adding kanamycin resistance to the cells); 2) flanking regions at both ends spanning tcpH and tcpA to allow double-homologous recombination; 3) the specific T7 RNA polymerase promoter sequence (black box stating T7 in Fig. 4A). This PCR fragment was obtained in two rounds of PCR as described earlier [3] and used as transforming DNA in a chitin-induced transformation assay [2]. The acceptor strain in this assay was ADctxAB-T7RNAP (T7RNAP + ). Transformants were selected on kanamycin-containing agar plates followed by the excision of the Kan R cassette using FLP recombination as previously described [3]. The resulting V. cholerae strain, ADctxAB-[P T7 ]-tcp-T7RNAP, was verified for the integration of the T7 RNA polymerase specific promoter sequence by PCR using purified genomic DNA as template (Fig. 4B).

T7 RNA polymerase-dependent expression of tcp genes under non-virulence inducing conditions
The newly created strain ADctxAB-[PT7]-tcp-T7RNAP ([P T7 ] + , T7RNAP + ) as well as the parental strains lacking either the T7 gene 1 (ADctxAB-[P T7 ]-tcp) or lacking the T7 specific promoter sequence (ADctxAB-T7RNAP) were grown under nonvirulence inducing conditions. After harvesting the cells, RNA was extracted and qRT-PCR was performed (Fig. 5). The expression of diverse VPI-1 genes located either upstream (tcpI and tcpH) of the T7 specific promoter sequence or downstream of the latter (tcpA,C,C,D,T,F, toxT, acfB,D; Fig. 5) was determined. As depicted in Fig. 5 all genes located downstream of P T7 were at least 100-fold induced compared to either parental strain ( Fig. 5B and C). In contrast the expression of genes located upstream of P T7 were similar to the expression measured for the parental strains and in the range of the expression that was also observed for a housekeeping gene (recA). Importantly, neither the T7 RNA polymerase nor the P T7 promoter alone was sufficient to induce tcp expression (Fig. 5D) in contrast to the strain containing both elements (Fig. 5B, C). We conclude that the T7 RNA polymerase/promoter system is able to drive tcp expression in V. cholerae.
Phenotypes associated with T7 RNA polymerasemediated tcp expression in V. cholerae Taylor et al. provided evidence that in vitro induction of TCP (under virulence-inducing conditions of V. cholerae biotype classical) resulted in autoagglutination of the bacteria [19]. Such autoagglutination led to the formation of macroscopic bacterial clumps and ''large aggregates that collect rapidly at the bottom of the culture tube'' [30]. Closer inspection of such clumps by SEM suggested that they are reminiscent of microcolonies formed upon intestinal colonization [30]. Furthermore, the authors of this study concluded that ''the ability of TCP mutants to participate in microcolonies correlates with the ability of those strains to colonize the infant mouse'' [30]. Consequently, we checked for autoagglutination of V. cholerae as an in vitro phenotype after P T7 -driven expression of the tcp cluster (Fig. 6A). Indeed, autoagglutination was confirmed and fully dependent on the presence of the T7 RNA polymerase and the T7 RNA polymerase specific promoter sequence (Fig. 6A). In contrast, the parental strains harboring only one of those elements did not autoagglutinate. Furthermore, no cellular clumps were visible in the absence of autoinducer (-IPTG) even though we provided evidence that low expression also occured under those conditions (Fig. 3). This is consistent with the insufficient expression of tcp upon artificial toxT expression as discussed above [29].
As agglutinated bacteria are thought to resemble microcolonies formed in vivo [30] the bacterial cultures were observed using light microscopy (Fig. 6B). Whereas the bacteria were uniformly distributed in those cultures that also appeared homogenously at the macroscopic scale (Fig. 6A) the bacterial aggregates of strain ADctxAB-[P T7 ]-tcp-T7RNAP grown in the presence of inducer were reminiscent of microcolonies (Fig. 6B) and very similar in appearance to previously published studies [32].
For visualization of the TCP pili using SEM (Fig. 7), the bacterial cells were grown as described above and as illustrated in Fig. 6. Subsequently, the bacteria were allowed to attach to a silicon wafer before being processed for SEM. It should be noted that the macroscopic clumps observed in Fig. 6A were partly flushed from the surface upon immersion of the silicon wafer into the fixative. This is consistent with the TCP pilus being involved in cell-to-cell interactions more so than for attachment to the silicon wafer. Nevertheless, enough bacteria were retained on the surface to allow visualization the TCP fibers (Fig. 7). Indeed, a plethora of TCP fibers were visible in the T7RNAP + and P T7 -carrying strain (Fig. 7A+B), whereas no such fibers were visible in a strain lacking the P T7 promoter upstream tcpA (Fig. 7C+D). Interestingly, Jude and Taylor have recently demonstrated that V. cholerae O1 El Tor derived TCP pili are thinner in width than classical TCP pili [32], which is in accordance with the thin TCP fibers we observed in our SEM images (Fig. 7A+B). Figure 7. Visualization of TCP fibers by scanning electron microscopy. V. cholerae cells were grown in rich medium as described for Fig. 6. At that stage bacteria were transferred to silicon wafers and processed for SEM. A representative image of V. cholerae strain ADctxAB-[P T7 ]-tcp-T7RNAP containing both the T7 RNA polymerase gene and P T7 promoter preceding the tcp cluster is shown in panel A (EHT = 2.00 kV, WD = 3.7 mm, Mag = 13.48 k X). The control strain lacking the P T7 promoter sequence upstream the tcp cluster is shown in panel C (EHT = 2.00 kV, WD = 3.8 mm, Mag = 24.91 k X). The white rectangles in panel A and C indicate the regions that are magnified in panels B and D, respectively. Scale bar = 1 mm. doi:10.1371/journal.pone.0053952.g007

Conclusions
In conclusion we successfully used the T7 RNA polymerase/ promoter system in V. cholerae. To our knowledge this is the first time that this system was utilized in this organism though it was already suggested in earlier studies that ''comparable T7 expression systems [to those in E. coli] can be developed in other types of cell'' [12]. The advantage of this system is that the T7 RNA polymerase is highly selective for transcription from own promoters. A disadvantage that we encountered here and which was already reported earlier is that the lacUV5 promoter is not completely tight and thus leads to low expression of the T7 RNA polymerase even in the absence of inducer [12]. However, this problem has been addressed by others and several options to either tighten the promoter (e.g. using Lac repressor variants and/or production of increased levels of the repressor) or to reduce basal activity of undesired T7 RNA polymerase [43] have been suggested. As a next step we will exchange the lacUV5 promoter preceeding the T7 gene 1 for the arabinose-inducible P BAD promoter [44], which we have used with great success in earlier studies [7]. Judson and Mekalanos also used the P BAD promoter in V. cholerae [45]. In their study P BAD was outward-facing from a mariner-based transposon and as such used to transcriptionally fuse the promoter to different neighboring genes within the transposon library [45]. Here, we did not use random transposon insertion but instead we demonstrated target-specific gene expression as the T7-specific promoter was integrated by natural transformation and thus in a site-directed manner.
The data provided here demonstrate that the strategy depicted in Fig. 1 is functional. Thus, the combination of T7 RNA polymerase and the integration of the T7 RNA polymerase specific promoter sequence using natural transformation and FLP recombination (TransFLP) seems to be a promising tool to artificially induce gene clusters in V. cholerae.

Supporting Information
Table S1 Primers used in this study. (DOCX)