Table 1 Bacterial strains used in this
study. Strain Relevant genotype Source or reference E. coli JM109 recA1 endA1gyrA96 thi-1 hsdR17 (r K – m K +) Stratagene supE44 relA1Δ(lac-proAB) [F' traD36 proAB lacI qZΔM15] E. coli BL21(DE3)pLysS F- ompT r – B m – B dcm gal tonA (DE3) pLysS (CmR) [46] E. coli EP314 W3110 Δ(lacIOPZYA) exa-1::Mu [19] dI1734 Km lac) in cadA] cadC1::Tn10 E. coli EP-CD4 E. coli EP314 lysP::Cm This work E. coli MG1655 K12 reference strain [47] E. coli MG1655ΔdsbA E. coli MG1655 ΔdsbA::Kan This work E. coli MG1655ΔdsbB E. coli MG1655 ΔdsbB::Kan This work E. coli MG1655ΔdsbC E. coli MG1655 ΔdsbC::Cm This work E. coli MG1655ΔdsbD E. coli MG1655 ΔdsbD::Kan This work E. coli MG1655ΔdsbG E. coli MG1655 ΔdsbG::Kan This work E. coli MG1655ΔccmG E. coli Nutlin-3a research buy MG1655 ΔccmG::Kan This work Table 2 Plasmids used in this study. Plasmid Relevant genotype Source or reference Aurora Kinase inhibitor pET16b Expression Crenolanib concentration vector, Apr Novagen pET16b-cadC cadC in pET16b [6] pET16b-cadC_C172A
Amino acid exchange C172A in cadC, This work cadC_C172A in pET16b pET16b-cadC_C208A cadC_C208A in pET16b This work pET16b-cadC_C272A cadC_C272A in pET16b This work pET16b-cadC_C172A,C208A cadC_C172A,C208A in pET16b This work pET16b-cadC_C172A,C272A cadC_C172A,C272A in pET16b This work pET16b-cadC_C208A,C272A cadC_C208A,C272A in pET16b This work pET16b-cadC_C172A,C208A,C272A cadC_C172A,C208A,C272A in pET16b This work pET16b-cadC_C208D,C272K cadC_C208D,C272K in pET16b This work pET16b-cadC_C208K,C272D cadC_C208K,C272D in pET16b This work pET16b-cadC_C172A,C208D,C272K cadC_C172A,C208D,C272K in pET16b This work pBAD33 Expression vector, Cmr [48] pBAD33-lysP lysP in pBAD33 Liothyronine Sodium [11] Site-directed mutants are designated as follows: The one letter code is used, followed by a number indicating
the position of the amino acid in wild-type CadC. The sequence is followed by a second letter denoting the amino acid replacement at this position. Generation of plasmids and strains All cadC derivatives were constructed by polymerase chain reaction (PCR) with mismatch primers either by single step or by two step PCR [41]. To facilitate construction, a cadC gene with two additional unique restriction sites was employed [11]. All site-specific mutations were directed by synthetic oligonucleotide primers containing the required nucleotide exchanges. PCR fragments were cloned into the expression vector pET16b with the restriction enzymes NdeI and BamHI so that all constructs carried the sequence encoding an N-terminal His-Tag of 10 histidine residues. E. coli EP-CD4, E. coli MG1655ΔdsbA, E. coli MG1655ΔdsbB, E. coli MG1655ΔdsbC, E. coli MG1655ΔdsbD, MG1655ΔdsbG and MG1655ΔccmG were constructed by deleting the genes lysP, dsbA, dsbB, dsbC, dsbD, dsbG and ccmG, respectively, via the Quick & Easy E.