Deletion of Helicobacter pylori vacuolating cytotoxin gene by introduction of directed mutagenesis

Figure 1 The strategy for disruption of vacA gene by directed mutagenesis. LA and RA were PCR-amplified from H pylori NCTC 11638 genomic DNA, and the kanamycin resistance gene, from pEGFP-N1. PCR products with different restriction sites on both sides were digested with corresponding endonucleases, and then cloned into pBluescript II SK digested with the same enzymes. Because there is an EcoR II site in RA, pLA and pkm^r were firstly joined together, resulting in pLK, which then was joined with pRA, resulting in pLKR. The plasmid pLKR was transformed into H. pyloi NCTC 11638, where the Km^r marked mutation was introduced into the genome by homologous recombination, resulting in the vacA^-Km^r mutant strain.