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

We performed the genetic screen using a pooled E. coli barcoded knockout strain library. To set up the screen, A 200 uL aliquot of frozen glycerol stocks of the library was grown overnight in 15 mL LB supplemented with 25 ug/mL chloramphenicol and grown overnight for 15 hours. The cells were washed twice in PBS, resuspended in M9, and diluted 1:50 in 40 mL M9 to grow for two additional hours. OD600 was then measured and the culture was normalized to OD600=0.1 in 60 mL M9. The library was then mixed 1:1 or 10:1 (toxic strain to knockout library ) with colibactin-producing bacteria or non-colibactin-producing bacteria (cultured overnight, washed, and grown for two hours as the library was) in 8 mL replicates, which were evenly spread across 16 wells in 96 deep-well plates (Eppendorf, cat# 2231000920). The co-cultures were pelleted and grown until collection at 8-, 24-, and 48-hours. At each time point, cultures were resuspended and each replicate was merged to a single tube for storage 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.