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

Ahani Azari, Ania; Zahraei Salehi, Taghi; Nayeri Fasaei, Bahar; Madadgar, Omid; Alebouyeh, Masoud

doi:10.22059/ijvm.2014.51890

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

Authors

Ania Ahani Azari ¹

Taghi Zahraei Salehi ¹

Bahar Nayeri Fasaei ¹

Omid Madadgar ¹

Masoud Alebouyeh ²

¹ Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

² Bacis and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

10.22059/ijvm.2014.51890

Abstract

BACKGROUND: Among all common techniques in sitedirected
mutagenesis, λ Red recombinase system has been
widely used to knock out chromosomal genes in bacteria. In this
method, there is always the risk of DNA Linear digestion by
host's restriction enzymes that leads to the low frequency of
recombination. OBJECTIVES:To overcome this, we constructed
a recombinant vector to disrupt phoP gene in Salmonella
typhimurium. METHODS: The SOEing PCR method and
restriction enzymes were used to construct the vector. RESULTS:
The resulting plasmid, pTAAZ92, contains a Kanamycin
cassette with two long homologous arms flanking of the phoP
gene. CONCLUSIONS: After electrotransformation of the
pTAAZ92 into the Salmonella typhimurium , the phoP gene is
replaced by the Kanamycin cassette through homologous
recombination. According to the high homology of the phoP
gene in many of Salmonella species the pTAAZ92 can be used to
disrupt the phoP gene in most of these species.

Keywords

gene disruption

Kanamycin cassette

Salmonella typhimurium

sitedirected mutagenesis