Donor E. coli strains were inoculated into 3 ml of TB media with Carb (100 μg/ml) and grown for approximately 16 hours at 37°C without shaking. An appropriate recipient MG1655 E. coli strain (CmR DA28102 or KanR fAYC002) with contrasting resistance markers was similarly grown, but with 50 μg/ml selecting antibiotic and shaking. Following growth, donor cultures were diluted 10× and regrown for 2 hours to enter the exponential phase. Recipient and exponential-phase donor cultures were pelleted at 2000 relative centrifugal force (rcf) for 10 min at 25°C and resuspended in M9CA media, equalizing OD600. At this point, aliquots of donor and recipient cultures were taken for quantification via agar plating and controls for outgrowth conjugation.

Donor and recipient cultures were then mixed in a 1:1 ratio for conjugation, distributed in 500-μl volumes, and incubated for 1 hour at room temperature while being exposed to antibiotic or control test conditions. Antibiotic test conditions were applied in 0.5×, 1×, and 2× multiples of IC50s determined for the primary MG1655 E. coli recipient. IC50 values for Carb, Cm, Kan, Ery, and Norf were as follows: 1.91, 1.92, 2.13, 20.20, and 0.05 μg/ml (9). These growth conditions were chosen to restrict growth during conjugation. After 1 hour, cell mixtures were vortexed to disrupt conjugation, pelleted, and resuspended in 500 μl of M9CA. The separated donor and recipient conjugation control aliquots were then mixed to track outgrowth conjugation.

The experimental and outgrowth conjugation control curves must be displaced in time; otherwise, ΔOD will not reach target OD600 thresholds. For this reason, all cell mixtures were diluted 150× in M9CA with dual antibiotic selection [Carb (100 μg/ml), Cm (70 μg/ml), and Kan (50 μg/ml)] according to the donor-recipient pairs and then distributed three times in 150-μl volumes onto a microtiter plate. Microtiter cultures were covered with 50 μl of mineral oil to prevent evaporation and grown in a plate reader for 24 hours at 37°C, shaking before each OD600 measurement. The 150× dilution was chosen on the basis of an estimated η of ~10−14 from previous growth-restricted E. coli measurements. For best results, T0 should be maximized while minimizing outgrowth η. Outgrowth dilutions and temperature may need to be adjusted on a strain-by-strain basis to optimize separation between experimental and control curves. For E. coli, the most common plasmids belong to the IncF family. Therefore, to maximize T0 with the E. coli library, we used rich, buffered TB media for optimal conjugative pili formation and exponential-phase donor cultures for activation of F plasmid transfer machinery (30). Conversely, recipients are kept in stationary phase for decreased motility or cell wall modifications thought to improve pili tip searching (9, 36).

Transconjugant growth curves were passed through a moving average filter in MATLAB to reduce noise. Time to specified OD600 threshold was found via linear interpolation. OD600 thresholds were typically chosen between 0.03 and 0.05 in early exponential phase to reduce background noise, maintain the cell density:OD relationship, and lessen postantibiotic effects (37). Within this range, changes in OD threshold had little effect on the correlation between τ and T0 (fig. S2B).

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