Mueller hinton 2 broth cation adjusted

Full-length icr^Mc and its catalytic domain (icr^Mccat; residues 235–578) plus full-length E. coli mcr-1 and its catalytic domain (mcr-1cat; residues 208–541) were ligated into the pGDP1 and pGDP2 vectors (in house derivatives of the pBR322 vector; New England BioLabs). These plasmids utilize constitutive promoters of differing strength; the stronger β-lactamase P_bla promoter (pGDP1) and the weaker P_lac promoter (pGDP2). Amino acid substitutions in icr^Mc were performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) and the pGDP1:icr^Mc construct. Constructs were confirmed by sequencing (Central MOBIX facility, McMaster University). Susceptibility testing (in triplicate, with two biological replicates) was performed with E. coli BW25113 cultured in Mueller-Hinton II broth (cation adjusted) (Becton, Dickinson and Co, Franklin Lakes NJ) using the broth microdilution method according to CLSI guidelines. Inoculum was prepared using the colony resuspension method, and plates were incubated with aeration at 37 °C for 18 h.