library_construction_protocol: The E. coli BW25113 transposon mutant library containing over 800,000 different mutants that was used in this study has recently described (Yasir et al., 2020). The transposon used to construct this library incorporates an outward-transcribing IPTG-inducible promoter. The library was used to inoculate parallel cultures of 5 mL LB broth (without salt) with approximately 10^7 cells. Cultures were grown in 6-well plates containing 40 sterile 5 mm glass beads per well (Sigma, 18406). Two replicates were set up, with or without 1 mM IPTG. Plates were incubated at 30 ˚C with light shaking for 48 hours. After 12, 24, and 48 hours of incubation, 2 mL of planktonic sample was collected from each culture and 70 beads were taken to constitute the biofilm sample. Beads were washed twice in sterile PBS and vortexed in tubes containing PBS to resuspend cells from the biofilm. Both planktonic and biofilm samples were centrifuged at 2100 x g to form pellets for DNA extraction.  To extract genomic DNA for sequencing, selected strains were grown O/N in a 96-deep-well plate in LB, at a final volume of 1.5 mL. Cells were recovered by centrifugation at 3,500 x g and were resuspended in 100 μL of lysis buffer (5 μg/mL lysozyme, 0.1 mg/mL RNAse in Tris-EDTA, pH 8) per well. The resuspended cells were then transferred in a new semi-skirted, low-bind PCR plate, secured with an adhesive seal and incubated at 37°C for 25 minutes. 10 μL of a lysis additive buffer (5% SDS, 1 mg/mL proteinase K, 1 mg/mL RNAse in Tris-EDTA, pH 8) was added in each well and the plate was sealed with PCR strip lids before being incubated at 65°C for 25 minutes. The plate was briefly centrifuged and 100 μL were moved to a new PCR plate. For the DNA isolation, 50 μL of DNA716 binding magnetic beads (KAPA Pure beads, Roche diagnostics) were added in each well and were incubated at room temperature for 5 minutes. The plate was then placed on a magnetic base and the supernatant was removed by pipetting. The beads were washed three times with 80% freshly-prepared ethanol. After removing the last wash, the beads were left to dry for 2 minutes before eluting the DNA. For the DNA elution, the plate was removed from the magnetic apparatus and 50 μL of Tris-Cl were added to each well. The beads were pulled using the magnetic apparatus and the isolated DNA was transferred to a new PCR plate. DNA concentration was determined using the Qubit ds DNA HS Assay kit (Q32851) following the manufacturer's instructions. A customised sequencing library was prepared to identify transposon insertions. DNA was tagmented using a MuSeek DNA fragment library preparation kit (ThermoFisher) and customised Tn5-i5 and i7 primers were used in PCR for 28 cycles (Yasir et al., 2020). DNA fragments of 300-500 bp were size selected using AMPure beads (Beckman Coulter).