Dig high prime labeling and detection kit

The C. difficile mutants were generated with the method of the ClosTron gene knock-out system, as described previously (Heap et al., 2007 (link)), combined with the TargeTron® Gene Knockout System Kit (Sigma-Aldrich). The intron DNA fragments were amplified with overlap PCR using specific retargeted primers (shown in Table S1) and then cloned into the HindIII and BsrGI restriction sites of pMTL007, using E. coli TOP10 as the host cells. After confirmation with PCR and DNA sequencing, the derived pMTL007 plasmids were each used to transform conjugative E. coli CA434, and then transferred into C. difficile 630Δerm by conjugation. The C. difficile transconjugants were selected in the presence of thiamphenicol, erythromycin, d-cycloserine, and cefoxitin. Once colonies appeared on the plates, the mutants were verified with PCR screening using the primers shown in Table S1. Southern blotting was performed with a DIG-High Prime Labeling and Detection Kit (Roche), according to the manufacturer's instructions. For the complementation experiments, each target gene, together with its native constitutive promoter, was cloned into pMTL84151. Using E. coli CA434 as the donor, the complementary plasmids were transferred individually into the C. difficile 630Δerm mutant strains to generate the strains used in this study (Table 1).

Genomic DNA was extracted from NXtc01 and tal mutants and digested with BamHI for 5 h. Digested DNA fragments were separated in 1.3% agarose gels in TAE buffer at 4^∘C, 80 V for 14–16 h and then transferred to Hybond N⁺ nylon membranes (Pall Corporation, NY, United States). The SphI fragment of the NXtc01 tal1 gene was used as a probe and labeled with digoxigenin (DIG) using the Dig-High Prime Labeling and Detection kit (Roche, Germany). DIG labeling, hybridization, washing, blocking, and detection were conducted as recommended by the manufacturer.