Terrific broth

E. coli BL21 (DE3) pREL containing the pET15b_hsmR plasmids were grown overnight in 5 mL of LB_carb50 at 37 °C. Cells were subcultured into Terrific broth (ThermoFisher Scientific) containing 50 μg/mL carbenicillin and grown to the mid-logarithmic phase of growth (0.5 abs measured at 600 nm) at 37 °C prior the addition of 1 mM isopropyl-1-thiol-D-galactopyranoside (IPTG). Growth was continued at 16 °C for 16 h. Cells were harvested by centrifugation (6000 × g for 10 min) and resuspended in 1 X PBS. Cells were lysed by passage through an EmulsiFlex homogenizer (Avestin) three times at 20,000 lb/in². The insoluble debris was removed by centrifugation at 40,000 × g for 1 h and the supernatant was filtered using a 0.22-μM-pore sizer filter. Filtered lysate was added to HisPur cobalt resin (ThermoFisher Scientific) and allowed to bind at 4 °C for 30 min prior to transfer to a gravity column. The column was washed with four column volumes of wash buffer (100 mM HEPES, 500 mM NaCl, pH 7.8) three times followed by 2 column volumes of elution buffer (100 mM HEPES, 500 mM NaCl, 200 mM imidazole, pH 7.8) twice. The hexahistidine tag was cleaved using the Thrombin Cleancleave kit (SigmaAldrich) by following the manufacturer’s instructions. After cleavage, buffer was exchanged utilizing overnight dialysis at 4 °C in 4 L of wash buffer (100 mM HEPES, 500 mM NaCl, pH 7.8).

C. difficile strains are of the 630Δerm background, and mutants were constructed using pyrE-based allele-coupled exchange.^{56 (link)} Strains were grown on brain heart infusion medium supplemented with 0.5% w/v yeast extract and 0.1% w/v l-cysteine (BHIS) with taurocholate (TCA; 0.1% w/v; 1.9 mM), thiamphenicol (10–15 μg/mL), kanamycin (50 μg/mL), or cefoxitin (8 μg/mL) as needed. C. difficile defined medium (CDDM)^{86 (link)} was supplemented with 5-fluoroorotic acid at 2 mg/mL and uracil at 5 μg/mL. Cultures were grown swirling at 37 °C under anaerobic conditions using a gas mixture containing 85% N₂, 5% CO₂ and 10% H₂. Escherichia coli strains were grown at 37 °C with shaking at 225 rpm in Luria–Bertani medium (LB) or at 20 °C with shaking at 225 rpm in autoinduction broth (Terrific broth [Thermo Fisher] with 0.5% glycerol, 0.05% glucose, and 0.1% α-lactose monohydrate). Media were supplemented with chloramphenicol (20 μg/mL), ampicillin (100 μg/mL), or kanamycin (30 μg/mL) as needed.