Plasmid Complementation and Gene Deletion Assays of E. coli Strains and Isolates

Plasmid complementation assays were performed using chemical competent cell preparations of E. coli BW25113, Keio collection gene deletion mutants (listed in Table 1), and CHRX1-3 isolates using the RbCl₂ protocol described in Green and Rogers (2013). ASKA collection strains AG1 (ME5305; pCA24N–), JW2343-AM (pMlaA–), JW5490-AM (pYghQ–), JW5248-AM (pMarR–), JW4276-AM (pFimE), JW3480-AM (pGadE), and JW5013-AM (pCdaR) were used to extract and purify their respective plasmid clones, using plasmid DNA extraction kits and isolation protocols from BioBasic Inc (ON, Canada). pCA23N(–) plasmids add an in-frame amino-terminal hexahistidine affinity tag to each cloned gene as described by Kitagawa et al. (2005). Plasmids were individually transformed into each E. coli strain listed above using the protocol described by Green and Rogers (2013) and transformants were selected and grown on LB medium with 30 μg/mL CM to maintain plasmid selection. Plasmids were re-isolated from each transformant to verify proper plasmid transformation. All transformants were examined using the same broth microdilution AST methods as described in sections above, at increasing CHX concentration ranges (0.5–16 μg/mL) to calculate any differences in MIC values between the transformants. All AST experiments were performed in triplicate for each transformed isolate or strain (Table 5).