Landscapes of collateral fitness effects of mutations in antibiotic resistance genes

A library of single-codon substitutions in either the NDM-1, CAT-I, or AadB antibiotic resistance genes was constructed using inverse PCR. The library was transformed into NEB 5-alpha F'Iq cells. We diluted uninduced, exponentially-growing cultures of the libraries mixed with wildtype versions of the antibiotic resistance genes into LB media containing IPTG. Cultures were allowed to grow for ~10 generations at 37C with a 32-fold dilution after ~5 generations of growth. We plasmid prepped and determined the allele frequencies of the generation 0 and generation 10 samples using Illumina MiSeq 2x300bp deep sequencing. Our growth competition is conducted in the absence of antibiotic so as to measure the collateral fitness effects of mutations in each of the antibiotic resistance proteins.