Flexible anaerobic chamber

Escherichia coli S-17 λ pir strains (13 (link)) were grown at 37°C in LB medium supplemented with carbenicillin 50 μg/mL. B. thetaiotaomicron VPI-5482 (ATCC 29148) derived strains were grown anaerobically at 37°C in liquid TYG medium (14 ). All anaerobic culturing was performed on brain-heart-infusion (BHI; Becton Dickinson) agar supplemented with 10% horse blood (Quad Five Co.). Cultures of bacterial gut communities and isolates for drug degradation assays were grown in Gut Microbiota Medium (GMM) (15 ). For selection, gentamicin 200 μg/mL, erythromycin 25 μg/mL, and/or 5-fluoro-2-deoxy-uridine (FUdR) 200 μg/mL were added as indicated. A flexible anaerobic chamber (Coy Laboratory Products) containing 20% CO₂, 10% H₂, and 70% N₂ was used for all anaerobic microbiology steps. Growth curves were performed as described in Supplementary Materials and Methods.

Minimal mucin media was made with 0.5% porcine mucin (M2378, Sigma), 100 mM KH₂PO₄ (pH 7.2), 15 mM NaCl, 8.5 mM (NH₄)₂SO₄, 4 mM L-cysteine, 1% vitamin K1-hemin solution (Becton Dickinson), 100 μM MgCl₂, 1.4 μM FeSO₄·7H₂O, 50 μM CaCl₂, 1% trace mineral supplement (ATCC), and 1% vitamin supplement (ATCC). Minimal M9 media was purchased (Teknova) and supplemented with 0.5% tryptone, plus or minus 0.25% porcine mucin (M2378, Sigma). Brain heart infusion medium (BHI) was purchased (Becton Dickinson) as agar or broth powder and made according to manufacturer’s instructions and supplemented with 1% vitamin K1-hemin solution (Becton Dickinson). BHI plus medium was supplemented with 5% heat inactivated fetal bovine serum, 1% vitamin K1-hemin solution (Becton Dickinson), 1% trace mineral supplement (ATCC), 1% vitamin supplement (ATCC), 3 mM D-(+)-cellubiose, 3 mM D-(+)-maltose, 6 mM D-(+)-fructose) and 4 mM L-cysteine. Media were filter sterilized using a Millipore Express filter unit (0.22-μm pore diameter) with the exception of porcine mucin, which was autoclaved in dH₂0 and added after sterile filtration. A flexible anaerobic chamber (Coy Laboratory Products) containing 20% CO₂, 5% H₂, and 75% N₂ and maintained at 37°C was used for all anaerobic microbiology steps.

All bacterial strains are listed in Table S5.
Bacteroides thetaiotaomicron VPI-5482 carrying a deletion in the tdk gene^{61 (link),62 (link)} was considered WT in all experiments and used for the generation of all barcoded and mutant strains. B. thetaiotaomicron strains were cultured in a flexible anaerobic chamber (Coy Laboratory Products) containing 20% CO₂, 10% H₂, and 70% N₂ at 37°C in liquid TYG medium and on brain heart infusion (BHI; Becton Dickinson) agar supplemented with 10% horse blood (Quad Five, Cat# 210-1000). Gut commensal isolates were grown in gut microbiota medium (GMM) ^{63 (link)}. For selection, gentamicin (200 μg/mL), erythromycin (25 μg/mL), and/or 5-fluoro-2-deoxy-uridine (FUdR) (200 μg/mL) were added as indicated.
E. coli S17-1 lambda pir and Citrobacter rodentium WT and Δtir strains were grown aerobically in LB medium at 37°C at 220 rpm. Ampicillin 100 μg/mL was used for selection of E. coli strains carrying pNBU2 and pExchange plasmids.