Real-time qPCR (RT-qPCR)

Pure cultures of B. dorei and B. dorei Δ5k-PUL33::p_compPartial1/2/3 growing on lactose, 3’-SL and 6’-SL (in replicates) were harvested at log phase (OD ~ 0.7) using the Direct-zol™ RNA Miniprep Plus kit (Zymo Research, R2071). Reverse transcription of the total RNA samples to cDNA was performed using SuperScript™ III Reverse Transcriptase (Invitrogen, 18080093). RT-qPCR reaction volumes were 4 μL 0.1 ng/μL DNA, 0.5 μL 10 μM forward primer (500 nM), 0.5 μL 10 μM reverse primer (500 nM), 5 μL iTaq Universal SYBR Green Supermix (BioRad, 1725124). For SusC only, the primer concentration was found to be optimal at 100 nM. The amplification program consisted of (1) 95 °C for 30 s, (2) 95 °C for 10 s (3) 60 °C for 30 s (4) repeat 2–3 for 39 times. The fluorescent products were detected at the last step of each cycle using a CFX96 instrument (BioRad). Primer efficiencies were optimized and calculated using a standard curve and relative changes in gene expression were calculated compared to the housekeeping gene rpsL¹⁰¹, with the efficiency-corrected ΔCq method¹⁰².