Sodium chenodeoxycholate

To determine if sodium-cholate, sodium-chenodeoxycholate, and ox-bile (all from Sigma-Aldrich) could induce porin switching in minimal media similar to that described previously (Mey et al., 2012 (link)), V. cholerae O1 El Tor N16961 wild type, ΔtoxR, ΔtoxS and ΔompU strains, as indicated, were grown overnight at 37 °C in LB media. The next day, the cultures were diluted 1:100 in minimal T-media (100 mM TRIS, 100 mM NaCl, 50 mM KCl, 20 mM NH₄Cl₂, 2 mM KH₂PO₄, 1 mM Na₂SO₄, 1 mM CaCl₂•2H₂O, 0.5 mM MgCl₂•6H₂O, pH 7.4) (Simon and Tessman, 1963 ) supplemented with 0.2% (wt/vol) sucrose, 20 μM FeSO₄, and 1x vitamin mix (10 μM of each: thiamine HCl, calcium pantothenate, p-aminobenzoic acid, p-hydroxybenzoic acid, 2, 3-dihydroxybenzoic acid), as described in http://www.genome.wisc.edu/resources/protocols/ezmedium.htm, containing either 0.3% ox-bile, 0.1% cholate, 0.01% CDC, or no compound as a control. The cultures were incubated for another 8 hours at 37 °C. The cultures were then pelleted and whole cell lysates were prepared. The protein concentrations of the lysates were determined using the BCA assay (Thermo Fisher Sci.). Equal amounts of protein were run on a precast 16% SDS-PAGE gel (Novex). The gels were stained with Colloidal Blue (Invitrogen) to visualize the porins.