Figure 5. PCR screening of double crossover candidate clones for complementation of the cwp84 gene in C. difficile 630 Δerm Δcwp84.

(A) Schematic diagram of the complementation of cwp84, with a single nucleotide change to base 2280 of cwp84 from a T to an A, without changing the corresponding valine amino acid residue and at the same time creating a ScaI site. The purpose of this single nucleotide change was to prove the occurrence of the complementation event. (B) PCR screening of candidate clones of the complemented cwp84 gene. Primers cwp84-F4 and cwp84-R4 anneal to the internal sequence of the knockout cassette and the downstream sequence of cwp84, respectively, resulting in a 1, 026 bp PCR product from double-crossover complemented clones and wild-type, while no PCR product is expected from Δcwp84 mutants. MW is a 2-Log DNA Ladder (NEB) molecular weight marker, WT is a wild-type C. difficile DNA control, and 1–3 are the candidate clones. All candidates 1 to 3 show the expected complemented 1, 026 bp band, thereby confirmed as cwp84 complemented clones, as seen in the wildtype control. (C) PCR products amplified using primers cwp84-F4 and cwp84-R4 from candidates clones and wildtype were analysed by RE digestion with ScaI. PCR products amplified from cwp84 complemented clones were cut into two fragments (786 and 240 bp), whereas PCR products amplified from the wildtype control did not.