Lab evolution of E. coli to three non antibiotic drugs

We evolved drug resistant bacteria using a serial transfer protocol. We started the experiment by picking 18 isolated colonies of the BW25113 strain and growing them overnight in M9 media. We inoculated the cultures in M9 media supplemented with drug at the IC50 concentration. We inoculated five wells for each drug and three wells for DMSO controls (a total of 18 wells). We grew the cultures at 37 C and 200 rpm shaking and diluted the cultures 1:200 into fresh media daily, either with or without drug. We increased the drug concentration by 1.5-fold every fourth passage.After completing 27 transfers we froze samples as 20% glycerol stocks at -80 C. We isolated individual clones from each frozen stock by streaking evolved populations on agar plates and expanding five individual colonies, each originating from a single cell, in liquid culture. We determined the drug response curve of all clones on their respective drugs and selected the most resistant clone from each population for whole genome sequencing through SeqCenter.Sample libraries were prepared using the Illumina DNA Prep kit and IDT 10bp UDI indices, and sequenced on an Illumina NextSeq 2000, producing 2x151bp reads. Demultiplexing, quality control and adapter trimming was performed with bcl-convert (v3.9.3).