E. coli targeted barcoded genetic screen in the presence and absence of colibactin-producing bacteria

A subset of 201 pooled (including 38 control strains), barcoded knockout strains were screened in co-cultures with both colibactin-producing bacteria and non-producing bacteria. To start the screen, individual strains were picked from glycerol stocks and grown overnight in 1 mL LB supplemented with 25 ug/mL chloramphenicol for 16 hours. The strains were pooled by combining 100 uL of knockout strain culture with 400 uL of each control knockout strain. We then washed 2 mL of this pooled culture twice in PBS before resuspending in M9, diluting 1:50 in 45 mL M9, and growing for three more hours at 37C. We then measured OD600 and diluted to OD600=0.1 and mixed the culture 10:1 (toxic strain to knockout library) with colibactin-producing bacteria or non-colibactin-producing bacteria (cultured overnight, washed, and grown for three hours as the library was). Co-cultures were mixed in 8 mL divided across 16 wells in a 96 deep-well plate (Eppendorf, cat# 2231000920). Co-cultures were pelleted, incubated for 24 hours, mixed and re-pelleted, before collection. All wells per replicate were combined, flash frozen and stored at -80C until DNA was extracted using Zymo Quick-DNA Midiprep Plus Kit (cat# D4075). We then amplified a region of ~350 bp around the barcode locus with custom forward and reverse primers using 2x KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Cat#KK2602) to produce the amplicon libraries for each sample. PCR products were purified with AMPure XP beads (Beckman Coulter, Cat#A63881) and run on a 2.5% agarose gel and extracted using ZR-96 ZymoClean Gel Recovery Kit (cat# D4021). We then sequenced using NextSeq 500/550 High Output Reagent Kit, 75-cycles (Illumina, Cat# 20024906) on Illumina NextSeq 500/550 device.