Figure 3

Identification and verification of Δ cpaAIR mutant colonies of Clostridium pasteurianum . A) Schematic diagram depicting primer annealing sites and expected PCR products of wild-type cells (left) and ΔcpaAIR mutant cells (right). Insertion of the Ll.ltrB intron into the cpaAIR gene leads to a 915 bp increase in size of the full-length PCR product generated using primers flanking the 176a intron insertion site (REN.Rv + REN.Fw). Both 5′ and 3′ gene-intron junction PCR products can be detected in ΔcpaAIR mutant cells using primer sets REN.Rv + ltrB.Rv and ltrB.Fw + REN.Fw, respectively. B) Colony PCR screening of gene disruption enrichment colonies for presence of intron insertion by amplification of the full-length product. Lane 1: marker; lane 2: no template control; lane 3: wild-type, non-recombinant C. pasteurianum colony; lanes 4 to 10: gene disruption enrichment colonies; lanes 4, 5, 7, and 8: positive colonies; lanes 6, 9, and 10: negative colonies. C) Further genomic verification of a single positive ΔcpaAIR mutant colony by amplification of all three PCR products depicted in Figure 3A (5′ junction, 3′ junction, and full product). A wild-type C. pasteurianum colony was included as a control for all three PCR primer sets. Lane 1: marker; lanes 2 to 4: wild-type colony; lanes 5 to 7: ΔcpaAIR mutant colony; lanes 2 and 5: 5′ junction; lanes 3 and 6: 3′ junction; lanes 4 and 7: full product.