The aim of this procedure is to genetically manipulate vio cray using this quick and deficient method, which is based on natural transformation and flip combination. This is accomplished by first preparing a proper chitin source as chitin is the natural inducer of transformation. During the second step, A DNA construct is generated using two rounds of PCR and their natural transformation is performed, allowing the plasmid PBR flip containing bacteria to take up the PCR fragment and incorporated into their genomes.
Next, a temperature upshift is used to induce the plasmid encoded lipase enzyme for removal of the flip recombination target or FRT site flanked antibiotic resistance cassette. In the final step, the bacteria are cured of the FLP encoding plasmid. Ultimately, PCR and sequencing are used to show that the genetic manipulation was successful.
The main advantage of this technique over existing methods, such as using suicide plasmids to genetically manipulate vire is that this method is very fast and efficient. Begin by weighing 50 to 80 milligram samples of chitin flakes in standard 1.5 milliliter plastic tubes, then keeping the lids of the tubes open, autoclave the flakes after the autoclave has called immediately close the lids and store the autoclave kit in flakes at room temperature at least six oligonucleotides are required to amplify the DNA regions of interest, as well as the FRT flanked antibiotic cassette by PCR. It is also recommended to include a pair of oligonucleotide primers outside of the inserted PCR fragment.
This allows checking for integration and correct excision of the FRT flanked antibiotic resistance cassette design, the number two, number three, number four, and number five, oligonucleotides with care. Primers two and five should contain at least 28 base pairs to be able to sufficiently an kneel to the genomic DNA and primers. Three and four should be constructed with an FRT can FRT containing plasmid as a template design primers two and five to allow extensive complimentary base pairing at the five prime end between primers two and three and primers four and five respectively.
Now for the first round of PCR, use oligonucleotides one and two to prepare the first of three simultaneous, but independent PCR reactions for amplification of the upstream region of the experimental region of interest of the custom designed constructs. In parallel, prepare the second PCR reaction using oligonucleotides three and four to amplify the FRT flanked antibiotic cassette. For example, in this experiment, the A PH gene encoding for canin resistance was used.
Concomitantly amplify the downstream DNA region by preparing the third PCR reaction using oligonucleotides five and six and genomic DNA as the template. Then start the PCR reaction for all three samples after purification of all three of the PCR fragments, use a mixture with equal amounts of all three fragments obtained in the first round as a template to perform a second round of PCR. This amplification will be catalyzed by oligonucleotides one and six.
The resulting PCR fragment will serve as the transforming DNA in the natural transformation experiments. After growing v cholera cells, aerobically enriched medium at 30 degrees Celsius. Harvest the bacteria by centrifugation at room temperature.
Next, transfer the bacterial culture onto the kite in flakes and incubate the cell suspension overnight at 30 degrees Celsius. Next, carefully mix at least 200 nanograms of the second round PCR derived fragments into the bacterial cell suspension. Taking care not to extensively detach the bacteria from the chitin surfaces, then incubate this solution at 30 degrees Celsius without movement for another day.
Now vortex, the culture vigorously for at least 30 seconds for bacteria that harbor the PBR flip plasmid spread 100 to 300 microliters of the cell solution onto double selective plates containing ampicillin. In addition to the other antibiotic in the plates, incubate the bacterial cell cultures at 30 degrees Celsius until colonies are visible. Then isolate the single transformants from the selective plates.
Begin this step by growing the bacteria on LB agar plates containing ampicillin at 37 degrees Celsius for 16 to 24 hours. As an optional step, change the temperature for two to three hours to 40 degrees Celsius as the expression of flip from the PBR flip plasmid is de repressed at higher temperatures. After eight more hours of incubation at 37 degrees Celsius, transfer the bacteria to fresh plates.
Now restrict the clones in parallel on antibiotic containing and antibiotic-free agar plates to test for antibiotic sensitivity and then isolate a single resulting antibiotic sensitive colony. Begin this step by growing the culture overnight under aerobic conditions at 30 degrees Celsius, enrich medium without antibiotics. The next morning grow a new culture for three to six hours by diluting the overnight culture at a one to 100 ratio in fresh antibiotic-free medium in the afternoon streak or plate dilution of the culture onto plain LB agar plates, and then incubate the plates for another eight to 16 hours at 30 degrees Celsius until the colonies are visible.
In this first experiment aimed at deleting the neighboring genes CTXA and CTXB. The oligonucleotides for the trans flip method were generated as just demonstrated the parental strain, the intermediate strain harboring the FRT flanked antibiotic resistance cassette instead of the original DNA locus of C-T-X-A-B and the final strain deleted for C-T-X-A-B and freed of the resistance cassette were all tested for their genotype wholesale PCR was performed for this experiment using primers that did not kneel to the transforming PCR fragment, but only to the chromosomal DNA. The PCR fragments were separated by standard aerogel electrophoresis to determine their size.
In lane one, the world type B cholera strain was located at the expected fragment size of 3, 143 base pairs. In lane two, the PCR fragment derived from strain delta C-T-X-A-B-F-R-T can. FRT was located at 3, 162 base pairs.
And in lane three, the delta ctx A B FFR TP CR fragment was located at 1, 769 base pairs as compared to the one KB ladder. In the second example in which a whole genomic island was removed, the Vibrio Pathogenicity island one or VPI one was chosen as the target for this experiment. The trans flip method was modified in two steps.
First, the integration of the kanamycin resistance gene was proceeded by a single FRT site at the start of PI one. Then a second round of natural transformation was performed, allowing the insertion of an additional antibiotic resistance marker, namely Gentamycin resistance followed by a second FRT site at the end of the pathogenicity island. The respective primer pairs are indicated above each gel picture and the trans FLP method was continued as just demonstrated.
The parental, intermediate and final strains were all verified by PCR of the purified genomic DNA in lane one. The PCR fragment of the wild type V cholera strain was run in lane two. The PCR fragment resulting from strain VPI one FRT can GM FRT was run.
And in lane three, the strain delta VPI one FRT derived PCR product was analyzed. Primer pairs are indicated above each gel picture. The final strain lack the whole VPI one.
In this third example, A T seven RNA polymerase dependent promoter sequence was integrated upstream with the Gene T fox, a major regulator of natural transformation using the trans flip method with a slight modification from the standard protocol. The T seven RNA dependent promoter consensus sequence was included as overhangs into oligonucleotides number four and number five, so that this DNA sequence was maintained within the chromosome. Even after the antibiotic resistance cassette was excised, the construct was then integrated into a T seven RNA polymerase containing V colorized strain.
In this strain, the expression of the T seven RNA polymerase gene was driven by the LAC UV five promoter to test for the T seven RNA polymerase dependent expression of T Fox Natural transformation assays were performed in LB medium. The parental strain lacking the T seven dependent promoter preceding the Gene T Fox is not naturally transformable under these conditions due to the lack of T FOX transcription in rich medium and in the absence of its natural inducer chitin. This phenotype was independent of whether the T seven RNA polymerase was artificially induced by IPTG or not.
However, the trans flip generated strain, which contains the T seven RNA polymerase dependent promoter upstream of the competence Gene T Fox, was indeed naturally transformable enrich medium due to the leakage of the LAC UV five promoter in LB medium. The produced T seven RNA polymerase already transcribed T FOX from the T seven RNA polymerase dependent promoter without induction by IPTG. This initiated natural competence and transformation confirming the functionality of the integrated T seven RNA polymerase dependent promoter sequence.
This phenotype was significantly enhanced by full induction of the T seven RNA polymerase due to addition of the inducer of the LAC UV five promoter IPTG. After watching this video, you should have a good understanding on how to genetically manipulate VI cholera using a combination of chitin induced natural transformation and flip recombination. Once mastered, this technique allows you to delete genes s or genomic islands, as well as to integrate DNA sequences in a site directed manner within only a few days.
