E. coli strains used in this study were engineered via Lambda red–mediated recombination (“red recombination”) (48). Parental strains were transformed with the helper plasmid pSIM5, and red recombination was performed using standard protocols (49). Strains were cured of pSIM5 (which harbors a temperature-sensitive origin of replication) by overnight growth at 37°C.

To generate point mutations or insert sequences without selective markers, we used a “scarless” chromosomal engineering technique—i.e., a genome-editing strategy that eliminates extraneous sequences such as antibiotic resistance cassettes (50). We modified a dual selection/counter-selection cassette in pKD45 (51) consisting of a kanamycin resistance marker and a toxin ccdB driven by a rhamnose-inducible promoter (PrhaB). This original cassette required counter-selection to be done on minimal M9 plates containing rhamnose because the presence of other carbon sources allowed cells to avoid activation of the rhamnose promoter and escape counter-selection. Cell growth was extremely slow on these plates, requiring almost two full days of incubation before colonies became visible. To overcome this limitation, we replaced the rhamnose-inducible promoter with the arabinose-inducible ParaB using standard molecular biology techniques and isothermal assembly (52) using the NEB HiFi DNA Assembly Master Mix (New England Biolabs). The resulting kmR-araBp-ccdB (“KAC”) cassette allowed efficient counter-selection on LB plates containing 10% arabinose. We also constructed a variant of this cassette with the gentamicin resistance (gmR-araBp-ccdB or “GAC”).

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