High-Throughput Screening of E. coli Filamentous Mutants

Mid-log cultures of E. coli JW0941–1/pBAD24 (vector only), JW0941–1/pLau80 (encoding FtsZ-YFP under P_BAD control) and the UTI89 gDNA expression library were diluted to A₆₀₀ = 0.05 and induced with 0.2% (w/v) L-arabinose. Incubation was continued until A₆₀₀ = 0.80. Culture samples were placed on ice and then analysed and sorted with an Aria II flow cytometer (BD Biosciences), as described previously [20 (link)]. Specifically, 500,000 events from the “filamentous” gate were initially collected (“yield sort”), and then the sample was re-sorted to obtain ~ 20,000 events of higher purity (“purity sort”). Cultures were analysed at an average of 37,000 events per second for the initial yield sort, and at 14,000 events per second for the purity sort. Purity sorted cells and 1 mL of the unsorted sample were used to inoculate 5 mL LB media containing ampicillin and 0.2% (w/v) D-glucose and grown overnight at 37 °C with shaking (~ 150 rpm). Plasmid DNA from both cultures was extracted and purified using the Isolate II Plasmid Mini Kit (Bioline).