Sequencing of Vibrio cholerae mutants.pdf

PCR amplification was performed using Phusion DNA polymerae (Thermo Scientific) according to manufacture's recommendation. In-frame deletion in V. cholerae was performed as descibed. Briefly, a 500-bp 5-prime flanking sequence of the gene, including several nucleotides of the coding region, was PCR amplified. The two PCR products were annealed at their overlapping region and amplified by PCR to produce a single DNA fragment, using outer primers. The resulting PCR product, lacking most of the coding sequence of the gene, was digested with XbaI enzyme and ligated into a similarly digested pCVD442 suicide plasmid. pCVD442 containing mutant construct was introduced into E. coli SM10.lambda pir by electroporation. The donor, E. coli SM10-lambda pir containing the plasmid pCVD442 containing mutant construct, was used for conjugal transfer to rifampin-resistant V. cholerae A1552. A mixture containing equal volumes of the donor and recipient in LB was incubated for 6 h at 30C. The exconjugants were selected by plating the suspension onto LB plates supplemented with 100 microgram/ml of rifampin and 100 microgram/ml of carbenicillin (Cb) at 30C. After selection of the desired tranconjugants on rifampin-plus-Cb plates, they were streaked on LB plates with 10 percent sucrose at 30C. Several colonies were purified from the plates, tested for Cb sensitivity, and then analyzed for the deletion using colony PCR and DNA sequencing. Sequencing data are described here.