Fig. 3.

Insertion of gusA into the genomic ldhD locus. A Schematic diagram illustrating gusA insertion. The substrate contained ~1-kb flanks located next to each other on the genome. Allelic replacement resulted in disruption of the ldhD gene and simultaneous insertion of the cat marker and gusA gene. In theory, any nonessential locus can be targeted. Primers ldhD-testA (c) and gus-testB (d) used for PCR testing are shown. B Inspection of potential mutants by PCR testing using primers c and d, as shown in A. Lane 1 shows DNA ladder and lane 2 the wild-type strain expected to generate an amplicon of ∼2 kb. Lanes 3–22 were tested colonies, with correct mutants expected to generate amplicons of ∼4.7 kb