TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Summary

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

Abstract

Several methods are available to manipulate bacterial chromosomes^1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons^1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene⁴. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)⁵ (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism⁶ and other representative of the genus Vibrio such as V. fischeri⁷. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region⁸. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.

This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences⁵. Several optimization steps concerning the initial protocol of chitin-induced natural transformation⁶ are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes⁸, the donation of PCR-derived DNA as transforming material⁹, and the addition of FLP-recombination target sites (FRT)⁵. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase¹⁰.

Protocol

The TransFLP method (Figure 1) is exemplified by three different approaches: I) the deletion of two adjacent genes encoding virulence determinants of V. cholerae (e.g., ΔctxAB); II) the removal of a pathogenicity island (e.g., ΔVPI-1); and III) the integration of a T7 RNA polymerase-dependent promoter sequence upstream of a gene-of-interest, which subsequently allows its artificial expression in the respective strain background (e.g., T7-tfoX) (Figure 2).

1. Preparation of Chitin Flakes

1. Weight 50-80 mg of chitin flakes in a standard 1.5 ml plastic tube. This can be prepared in bulk. Chitin flakes are commercially available from Sigma (cat. number C9213).
2. Autoclave the flakes keeping the lids of the tubes open.
3. Immediately close the lids after the autoclave has cooled down.
4. Store autoclaved chitin flakes at room temperature.

2. Optional: Artificial Transformation of V. cholerae with Flp Recombinase-encoding Plasmid

1. Prepare electrocompetent V. cholerae cells using standard methods¹¹. Store aliquots of competent cells at -80 °C.
2. Add 1-2 μl of a regular mini-preparation of plasmid pBR-flp⁵ to the electrocompetent V. cholerae cells and transfer the mixture into an electroporation cuvette (0.2 cm gap width).
3. Apply a pulse at 1.6 kV.
4. Add 0.9 ml SOC medium, gently transfer cell to a standard 14 ml tube. Incubate non-moving for 2.5 to 3 hr at 30 °C.
5. Plate 100 μl and 300 μl on LB plates containing 100 μg/ml ampicillin and incubate at 30 °C overnight.
6. Purify single clone and store as glycerol stock for future TransFLP experiments.

3. Oligonucleotide Design and Polymerase Chain Reaction

1. At least six oligonucleotides are required to amplify the DNA region(s) of interest as well as the FRT-flanked antibiotic cassette by PCR (Figure 3). It is recommended to also include a pair of oligonucleotides priming outside the inserted PCR fragment. This allows checking for integration and correct excision of the FRT-flanked antibiotic resistance cassette (e.g. depicted as 'chk-up' and 'chk-down' primers in Figures 3 to 5).
2. Design the oligonucleotides #2, #3, #4, and #5 with care. They should be able to sufficiently anneal (e.g. ~28 bp) to the template DNA. The template DNA is genomic DNA (gDNA) for primers #2 and #5 and FRT-Kan-FRT-containing plasmid DNA such as pROD17¹² and pBR-FRT-KAN-FRT (this study) for primers #3 and #4 (Figure 3A).
3. Design the primers #2 to #5 allowing extensive base-pairing at their 5'-end between #2 / #3 and #4 / #5, respectively (Figure 3A inset). Two rounds of PCR follow.
4. Perform three independent PCR reactions as a 1^st round of PCR. The first one will amplify the upstream region of the gene / DNA region-of-interest with the aid of oligonucleotides #1 and #2. The PCR should result in a fragment of at least 500 bp in length to allow homologous recombination within the cells. Shorter fragments (~250 bp) can be sufficient⁸ but are not recommended.
5. In parallel perform the second PCR, which amplifies the FRT-flanked antibiotic cassette. For the examples provided here we mainly used aph (kan^R). Use oligonucleotides #3 and #4 for this reaction (Figure 3A).
6. Concomitantly, amplify the downstream DNA region by PCR (third sample). The PCR fragment should also be at least 500 bp in length. Perform the PCR with gDNA as template and oligonucleotides #5 and #6 (Figure 3A).
7. After purification of all three PCR fragments, perform the 2^nd round of PCR. Use an equal mixture of all three fragments obtained in the first round as template. The amplification is catalyzed by oligonucleotides #1 and #6. The resulting PCR fragment (Figure 3B) serves as transforming DNA in the natural transformation experiment (Figure 1).

4. Chitin-induced Natural Transformation

1. Grow V. cholerae cells aerobically in rich medium at 30 °C until they reach an optical density at 600 nm of approximately 0.5.
2. Harvest the bacteria by centrifugation. Wash pellet once in defined artificial medium (DASW⁶) before resuspending the cells in 2 volumes of DASW. Add 1 ml of culture to 50-80 mg of sterile chitin flakes (explained under point 1). If bacteria harbor plasmid pBR-flp (optional point 2), add ampicillin (50 μg/ml) to the culture. Incubate at 30 °C for 16-24 hr without movement.
3. Add ≥ 200 ng of the 2^nd-round PCR-derived fragment (explained under point 3). Mix carefully without extensively detaching the bacteria from the chitin surfaces. Incubate at 30 °C for 24 hr without movement.
4. Vortex the culture extensively for ≥ 30 sec. Spread 100-300 μl on selective LB medium plates (e.g. kanamycin or gentamicin-containing LB plates for examples provided here). Use double-selective plates if the bacteria harbor the plasmid pBR-flp by adding ampicillin concomitantly to the other antibiotics. Incubate plates at 30 °C for 16-24 hr or until colonies are visible.
5. Isolate single transformants from selective plates. Such transformants have replaced the original chromosomal locus by the PCR fragment due to a double crossover event. These strains can be directly used for further experiments in case the removal of the antibiotic cassette is not necessary.

5. Removal of Selective Cassette(s) by Flp-recombination

1. Artificially transform your transformants with plasmid pBR-flp as described under point 2 if not done before the natural transformation assay.
2. Grow the bacteria on LB agar plates containing ampicillin at 37 °C for 16-24 hr. Options: change the temperature in between for 2-3 hr to 40 °C as expression of flp from plasmid pBR-flp is de-repressed at higher temperatures^5,10; transfer bacteria to fresh plates after approx. 8 hr of incubation.
3. Test for antibiotic-sensitivity (demonstrated here for kanamycin; Figure 1) by restreaking the clones in parallel on antibiotic-containing and antibiotic-free agar plates. Isolate single sensitive colonies. Freeze ampicillin-resistant clone as glycerol stock if you intend to further modify the strain with other deletion / insertion using TransFLP.

6. Plasmid Curing

1. Grow culture overnight under aerobic condition and at 30 °C. Use rich medium without the addition of any antibiotic.
2. Optional: Grow fresh culture for 3-6 hr by diluting the overnight culture 1:100 in fresh antibiotic-free medium.
3. Plate or streak dilution(s) of culture on plain LB agar plate(s) and incubate at 30 °C for 8-16 hr (until colonies are visible).
4. Test for ampicillin-sensitivity of the clones by restreaking the clones in parallel on antibiotic-containing and antibiotic-free agar plates (Figure 1). Freeze ampicillin-sensitive strain as glycerol stock.

Results

Representative results of the three examples are shown in Figures 4 to 6. The first approach aimed at deleting the neighboring genes ctxA and ctxB. Together they encode the major virulence factor of V. cholerae, cholera toxin. We designed oligonucleotides for the TransFLP method as described above and according to Figure 3. The parental strain, the intermediate strain harboring the FRT-flanked antibiotic resistance cassette at the original DNA locus of ctxA...

Discussion

The TransFLP method described above and elsewhere⁵ has been extensively used in our laboratory. The kind of genetic manipulations that are feasible include amongst others: deletion of single genes and gene clusters, deletion of genomic islands (e.g., VPI-1), insertion of sequences upstream of a gene of interest (e.g., promoter sequences) and insertion of sequences at the end of a specific gene (e.g., encoding affinity tags).

An import prerequisite for Tran...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chitin flakes	Sigma	C9213	Autoclaving required
OPTIONAL: Micropulser (in case antibiotic cassette should be excised by Flp recombinase encoded on pBR-flp)	Biorad	165-2100	Or comparable electroporators