Figure 3

The hpdBCA operon and verification of mutant construction. The hpdBCA operon with insertion sites for the targeted ClosTron mutagenesis, the number refers to the insertion site (bp) and the s/a refers to sense/antisense orientation of the ClosTron insert. B) PCR screen of the mutants (M = mutant; W = wild type; P = plasmid and "-" negative control). Three PCR screens were performed, gene specific forward and reverse primers, intron specific with gene specific primers, and RAM specific primers (Heap et al., 2007). C) Southern blot using a probe specific to the inserted intron. HindIII digests were performed on DNA from M = mutant; W = wild type; P = plasmid. The strains and primer sets are indicated on each figure and in tables 1 and 2. The marker sizes are indicated on the figure and the expected band sizes are as follows for 630Δerm: hpdB 2.8Kb; hpdC 3.4 kb and for R20291: hpdA - 6.3 kb; hpdC - 3.4 kb.