Cre/loxP mediated excision of inserted gene fragment from Salmonella typhi genome

Site-specific DNA recombinant system Cre/loxP from bacteriophage P1 has been developed as a novel tool for DNA manipulation, and used successfully both in vitro and in vivo. Here a protocol for effective excision of neo gene located on chromosome of Salmonella have been developed. In order to establish a tetracycline-induced expression system in the attenuated Salmonella typhi CVD908 strain, a fused DNA fragment consisting of genes of tetracycline repressor (tetR) as well as neo gene flaked by two loxP sequences in same orientation has been integrated into a defined Delta aro C locus of CVD908 strain via homologous recombination. To excise the neo gene from the locus, the suicide plasmid pJG9/Cre expressing Cre recombinase under the control of tetracycline response promoter PL,tal was constructed and electropolated into the CVD908 strain. The expression of the Cre recombinase induced by anhydrotetracycline successfully excised the neo gene. Since the suicide plasmid contains SacB gene encoding an enzyme that is lethal to G- bacteria in the presence of sucrose, growing of the bacteria in a medium containing 10% sucrose cured the pJG9/Cre plasmid. Both antibiotic and PCR identification demonstrated the successful excision.