RNA Extraction

Single colonies of transconjugants from the first conjugation round were grown in LB broth with shaking (125 rpm) overnight at 37°C. The cultures were diluted 1000-fold and grown with and without antibiotics to OD₆₀₀ = 0.5. The antibiotic concentration was 1/2 MIC. A FastPrep cell disrupter system (Qbiogene, Illkirch, France) and RNeasy Mini Kit (Qiagen, Sollentuna, Sweden) were used to extract total RNA by mechanical disruption. Quantity of the extracted RNA was determined by A260 measurements and purity by A260/280 ratio measurements using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Hvidovre, Denmark). RNA (1 μg) samples were purified by DNA digestion using TURBO^TM DNase kit (2 U/μL) (Ambion, Life Technologies, Nærum, Denmark) to remove contaminating genomic DNA.