One milliliter of overnight culture was washed (centrifuged for 5 min at 13,000 rpm, discarded the supernatant, and resuspended in 1 ml fresh M9) and diluted 1000-fold in fresh M9. The appropriate amount of cefotaxime (Sigma) was added to achieve 0, 1, 10, and 100 μg/ml. The cultures were incubated for 3 hours at 30°C. For each culture, 1 ml was kept on ice to preserve the population density at the 3-hour time point. Two milliliters was spun down (5 min, 13,000 rpm) and resuspended in 2 ml of water before being sonicated (20 amp, duration of 1 min at 4°C) to release periplasmic Bla. The sonicated culture was diluted 10-fold in water and then treated with 0 or 1 μM fluorocillin. A 96-well plate was loaded with 200 μl of each culture (whole cells), sonicated with and without fluorocillin, and topped with 50 μl of mineral oil. The plate was loaded into a Tecan Infinite M200 PRO microplate reader (chamber temperature maintained at 25°C), and OD600 and green fluorescent protein (GFP) readings were measured every 10 min for 1.5 hours. The GFP measurements of the sonicated samples were plotted over time, and the slope was calculated. The slope was normalized by the relevant culture’s OD measurement. A one-way ANOVA was used to determine any significant differences between the conditions.

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