Construction of a recombinant vector for site-directed mutagenesis in Salmonella typhimurium

Abstract

BACKGROUND: Among all common techniques in sitedirectedmutagenesis, λ Red recombinase system has beenwidely used to knock out chromosomal genes in bacteria. In thismethod, there is always the risk of DNA Linear digestion byhost's restriction enzymes that leads to the low frequency ofrecombination. OBJECTIVES:To overcome this, we constructeda recombinant vector to disrupt phoP gene in Salmonellatyphimurium. METHODS: The SOEing PCR method andrestriction enzymes were used to construct the vector. RESULTS:The resulting plasmid, pTAAZ92, contains a Kanamycincassette with two long homologous arms flanking of the phoPgene. CONCLUSIONS: After electrotransformation of thepTAAZ92 into the Salmonella typhimurium , the phoP gene isreplaced by the Kanamycin cassette through homologousrecombination. According to the high homology of the phoPgene in many of Salmonella species the pTAAZ92 can be used todisrupt the phoP gene in most of these species.