Cloning and mutagenesis of BrxR protein

PCR amplicons and plasmids were purified using Monarch DNA kits (NEB). PCR, restriction digests, ligations, transformations and agarose gel electrophoresis were performed using standard molecular biology techniques. Constructed plasmids were confirmed via sequencing with an Abi 3370 DNA sequencer. The pSAT1-LIC-brxR⁺ expression construct adds a cleavable N-terminal His₆-SUMO tag. Primers TRB878 and TRB879 were used to amplify brxR from pEFER (gene pEFER_0020) for insertion into pSAT1-LIC (38 (link)) to produce pTRB446 via Ligation Independent Cloning (LIC) (Supplementary Table S1). Primers TRB876 and TRB877 were used to amplify brxR from pEFER which was inserted into pBAD30 (39 (link)) to produce pBAD30-his₆-brxR (Supplementary Table S1). Primers TRB1987 and TRB1988 were used to perform QuikChange (Invitrogen) mutagenesis to produce pBAD30-his₆-brxR-R17A (Supplementary Table S1).