Cloning in pBAD. For amplification of 285 bp ORF2 encoding toxin Xn-relE gene from the genomic DNA primer 2 with PstI site at 5' end and primer 7 with HindlH site at 3' end were used.
PCR amplified product and pBAD expression vector were digested with PstI and HindIII restriction enzymes and ligated producing pJSL4.
coli Top 10 strains were selected for the endogenous toxicity assay and all the constructs of pBAD plasmid were transformed in E.
In the endogenous toxic assay, control strain JSL containing pBAD vector alone was considered as 100% based on the growth profile after induction with arabinose when compared with arabinose-induced JSL3 strain containing Xn-relE toxin only as well as with JSL4 strain with full operon (XnrelE + Xn-relEAT).
The expression of the two genes in the strain which contained pBAD as control was similar to [DELTA]ygaE strain (Figures 2(a) and 2(b)).
Strains or plasmid Relevant characteristics Strains Salmonella enterica Wild-type strain; [z66.sup.+] serovar Typhi GIFU10007 [DELTA]ygaE GIFU10007; [z66.sup.+] [DELTA]ygaE (pBAD) [DELTA]ygaE harboring pBAD plasmid; [DELTA]ygaE (pBADygaE) [DELTA]ygaE harboring pBAD-ygaE recombinant plasmid; E.