Transcriptional profiling of Escherichia coli after addition of CO gas to aerobically and anaerobically growing cells. Escherichia coli

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 CO gas and over a time course of 2.5, 5, 10, 20, 40 and 80 min exposure to CO gas 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. Overall design: Time course experiment with samples taken immediately prior to or 2.5, 5, 10, 20, 40 or 80 minutes after addition of CO gas 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 three biological repeats were perfomed for the anaerobic condition with 2 technical (dye swap) repeats per biological repeat.