Quantitative Real-Time PCR Protocol

Oligonucleotides for quantitative real-time PCR (sequences are listed in Supplementary Table IV) were designed using Integrated DNA Technologies. Total RNA of 500 ng was reverse transcribed using the iScript complementary DNA (cDNA) synthesis kit (Bio-Rad). The reaction mix contained 5 μl of SsoAdvanced Universal Probes Supermix (Bio-Rad), 0.5 μl primer and probe corresponding to 250 nM primers and 125 nM probe (20 × stock) and 0.5 μl of cDNA. qPCR measurements were performed using a CFX96 real-time PCR machine (Bio-Rad). The relative expression levels were determined by a 2^−ΔΔG method. Each sample was normalized by the cycle threshold geometric mean using reference genes rrsA and cysG59 (link). Biological replicates consisted of three E. coli cultures exposed to equivalent inducer concentrations (0 or 5 ng ml⁻¹). Three qPCR technical replicates were performed and averaged for each sample.