Figure 3.

Influence of direct repeat length upon markerless mutagenesis efficiency. (A) Four separate 156 bp deletion constructs were created using IFDC3 and the previously described DR-CIMM methodology. The constructs only varied in the sizes of the inserted direct repeats after the IFDC3 cassette. Shown here are the results obtained from the final 4-CP negative selection step. The strains from top to bottom are: DR25 (25 bp direct repeats), DR100 (100 bp direct repeats), DR200 (200 bp direct repeats), and DR400 (400 bp direct repeats). (B) The number of markerless mutant clones obtained using a range of direct repeat lengths was compared. The data are presented as the means ± standard deviations from three independent experiments. ^*P < 0.05 and ^**P < 0.01 as determined by unpaired two-tailed t-test. (C) 4 CFU from the DR100, DR200, and DR400 constructs were randomly selected from the 4-CP plates and PCR-amplified to compare their genotypes. All clones exhibited the expected 156 bp deletion, resulting in smaller PCR amplicons relative to the parent wild-type (+).