FIG. 5.

Disruption of the chromosomal yiaE gene of E. coli. (A) Structure of pHD-Km. A BamHI site was generated in the middle of the yiaE gene, and a 1.4-kb BamHI fragment containing the kanamycin resistance gene was inserted at the BamHI site of pHD-Bam. The resulting plasmid, pHD-Km, was linearized with ScaI before P1 transduction. (B) Confirmation of correct disruption of the yiaE gene by PCR. PCR with primers 2KRA-5′ and 2KRA-3′ (Fig. 2) was done with chromosomal DNAs from E. coli W3110 and its mutant strain as templates. Lane 1, 1-kb ladder; lane 2, PCR product with W3110 genomic DNA as a template; lane 3, PCR product with W3110 (yiaE::Km) genomic DNA as a template; lane 4, BamHI-digested PCR product with W3110 (yiaE::Km) genomic DNA; lane 5, pUC4K digested with BamHI.