Transcriptional profiling of Escherichia coli after addition of CORM-401 to aerobically and anaerobically growing cells

Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in Evans media containing 2 mM glucose as the sole and limiting source of energy and carbon. The working volume was 200 ml, and the dilution rate 0.2 h-1. For aerobic growth, the air-flow rate was 0.1 l/min, and the dissolved oxygen tension was maintained at ~7% air saturation by measuring oxygen dissolved in the culture using a Broadley James D140 OxyProbe® electrode. For anaerobic growth, cells are grown on 100 % nitrogen gas bubbled at 0.1 l/min Cells were grown as above to steady-state, At steady-state, CO gas was bubbled through the culture at 0.1L/min. Samples were taken immediately prior to the addition of CORM-401 and over a time course of 2.5, 5, 10, 20, 40 and 80 min exposure to CORM-401 for subsequent analysis using microarrays. Cells were harvested directly into phenol:ethanol to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers. Time course experiment with samples taken immediately prior to or 2.5, 5, 10, 20, 40 or 80 minutes after addition of CORM-401 under either 0 or 100 % perceived aerobiosis. 2 biological repeats were performed for the aerobic condition with 2 technical (dye swap) repeats per biological repeat, and two biological repeats were perfomed for the anaerobic condition with 2 technical (dye swap) repeats per biological repeat.