Fitness was measured as described in Mehlhoff et al. (Mehlhoff et al. 2020 (link)) using a growth competition experiment. Our experiment differed only at the start. Frozen library stocks and wild-type cells were thawed on ice before 50 μl was added to 100 ml media containing 50 μg/ml spectinomycin and 2% w/v glucose in a 500 ml unbaffled shake flask. The cultures were incubated with shaking for 16 h at 37° C. The wild-type and library cultures were then diluted to an OD of 0.050 in 100 ml of media and mixed at a ratio of 2:98 based on volume in a fresh unbaffled flask. The cultures were incubated at 37° C with shaking until the OD was about 0.5 and the remainder of the growth competition followed as described in previous work. In short, the exponentially growing library cultures were induced with IPTG and allowed to grow for approximately ten generations with a single dilution at about five generations. Plasmid DNA was collected before induction and after ten generations of growth. Custom adapters were added to the plasmid DNA by PCR. Adapters were designed to be compatible with the Illumina platform and contained barcodes for identification of each timepoint and sample. Proper DNA size of the PCR products was validated by agarose electrophoresis gel before the PCR products were pooled and submitted for Illumina MiSeq (2 × 300 bp reads) at the Single Cell and Transcriptomics Core facility at Johns Hopkins University.