Figure 1

Overall design scheme for making site-directed, mutations by inserting foreign DNA into S. enteritidis genome; Step 1: Overlapping extension PCR is used to construct PCR-A product consisting of the I-SceI recognition sequence and Km^r gene flanked by homologous regions upstream and downstream to the insertion site in loop 9 of lamB in Salmonella enteritidis. Step 2: PCR-A is introduced into the chromosome by electroporation and Red recombinase-mediated, homologous recombination. Step 3: The intermediate construct is isolated by selection on Km plates. Step 4: Overlapping extension PCR is used to construct PCR-B, with a foreign epitope sequence flanked by lamB regions as in PCR-A. Step 5: Co-electroporation of pBC-I-SceI and PCR-B. Here PCR-B is integrated into the genome by homologous recombination to replace the I-SceI recognition sequence and Km^r gene. Step 6: S. enteritidis containing the foreign epitope sequence is isolated by a counter-selection technique utilizing the pBC-I-SceI plasmid which expresses the I-SceI enzyme and Cm^r gene.