Quikchange 2 mutagenesis protocol

Acetyl-CoA was purchased from CoALA Biosciences (Austin, TX (12) (link). To reclone SaPC into the pTXB1 vector (New England Biolabs, Ipswich, MA), the gene encoding SaPC in pET-27b was PCR amplified using forward (5'-GCC ATA TGA AAC AAA TAA AAA AG -3') and reverse primers (5'-CGT GAT GCA GTT AGT TGC TTT TTC AAT TTC G -3'). The PCR amplicon was subjected to restriction digestion with NdeI and SapI prior to ligation into a gel-purified pTXB1 vector digested with NdeI and SapI. WT SaPC pTXB1 was fully sequenced and was found to include a PCR-introduced G571S mutation. The mutation was corrected back to a glycine at residue 571 by mutagenesis using the whole-plasmid PCR technique according to the Quikchange II mutagenesis protocol from Agilent Technologies, Inc. (Santa Clara, CA), with forward primer 5'-GCG GAC GTA TTT AAA GAT GGT TTC TCA CTA G -3' and reverse primer 5'-CTA GTG AGA AAC CAT CTT TAA ATA CGT CCG C -3'. The complete gene sequence of WT SaPC in the pTXB1 vector was confirmed by DNA sequencing.

A pET30b-based expression plasmid containing E. coli BamA POTRA5 and transmembrane domain sequences (Ni et al. 2014) , blue), HdBamA (PDB 4K3C (Noinaj et al. 2013) , pink), NgBamA (PDB 4K3B (Noinaj et al. 2013) , green) and EcTamA (PDB 4C00 (Gruss et al. 2013 ), orange). The loop L6 insertion, corresponding to the locally dynamic residues, is highlighted in blue (residues 344-810) with C-terminal His 6 tag was obtained from Monika Schu ¨tz (University of Tu ¨bingen). The BamA transmembrane domain (residues 421-810) was cloned with an N-terminal His 6 -tag into pET15b, using NdeI and XmaI restriction enzymes. The QuikChange II mutagenesis protocol (Agilent Technologies Inc) was used to introduce the mutations C690S and C700S into BamA.