Construction of Gene Deletion Mutants in Yersinia pestis
The λ Red system is a simple method for disrupting chromosomal or plasmid genes using polymerase chain reaction products in Escherichia coli and Salmonella. Here we describe a method of generating gene deletion mutations of Yersinia pestis using the λ Red recombination system. In this procedure, the gam, bet, and exo genes, which are necessary for homologous recombination, are placed under the control of the L-arabinose-inducible ParaB promoter in plasmid pKD46. Then, L-arabinose is added to induce the expression of the Gam, Bet, and Exo proteins. Then, a linear DNA fragment carrying an antibiotic resistance gene flanked by short stretches (30–50 nt) of DNA homologous to regions flanking the deletion site is prepared using the polymerase chain reaction. Then, the DNA fragment is electroporated into Y. pestis cells carrying pKD46. With the help of the Red recombinase, the DNA fragment is recombined into the homologous region on the chromosome. Thus, the target gene in the chromosome is replaced by an antibiotic resistance cassette. Cells carrying the desired deletion are selected on a medium containing the corresponding antibiotics.
Key wordsGene deletion Mutant pKD46 Red recombinase Yersinia pestis
- 3.Yin J, Wang H, Li R, Ravichandran V, Bian X, Li A, Tu Q, Francis Stewart A, Fu J, Zhang Y (2017) A practical guide to Recombineering in Photorhabdus and Xenorhabdus. Curr Top Microbiol ImmunolGoogle Scholar