Fig. 2

PCR validation of DFCT9, an incA insertion mutant created using the GII(aadA) spectinomycin-selection cassette. EBs were transformed with pDFTT3aadA and serially passaged with spectinomycin selection. Mutants were plaque purified and a single plaque was expanded for PCR and phenotype analysis (shown in Figs. 5, 6). The insertion locus map is shown in panel a (DFCT3 is from [19] ) with the intron highlighted in red. The intron is inserted in a sense orientation to incA. The GII(aadA) intron in vector pDFTT3aadA was used to create the incA mutation in both DFCT9 and DFCT16 (Fig. 4). Expected PCR product sizes are listed in panel b. The primers used and reaction descriptions are listed in Table 1. PCR products were run on 0.8 % agarose gels, stained with ethidium bromide, and visualized with a UV light source. Molecular weight markers (kbp) are shown to the left of each gel. Images were inverted to improve contrast. PCR results are shown for the wild type strain, DFCT9, and pDFTT3aadA in panels c, d, and e, respectively