Project name: Escherichia coli

Description: Escherichia coli is a metabolically versatile bacterium that is able to grow in the presence and absence of oxygen. Here, the process of adaptation was investigated by determining changes in transcript profiles when aerobic steady-state cultures were depleted of air.Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow 1000 fermentation vessels (1.8 l capacity) with culture agitation speed constant at 300 rpm and the temperature maintained at 37 °C. Oxygen levels were monitored using galvanic oxygen electrodes while the pH was maintained at 7.2 ±0.2 by automatic titration with sterile KOH. Evans defined medium was used as the growth medium with glucose (15 mM) as the carbon source with the dilution rate being 0.2 h-1. Aerobic cultures were maintained by sparging the chemostat with air (0.4 l min-1). The switch to micro-aerobic conditions was achieved by switching off the air sparging the culture. After a period of 5, 10, 15 and 60 min exposure to air, cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA before total RNA purification using Qiagen’s Rneasy Midi kit as recommended by manufacturer’s instructions.Keywords: time-course, oxygen-depletionOverall design: Analysis used aerobic steady state RNA as control samples for comparison to the experimental samples taken at 5, 10, 15 and 60 min. Indirect comparisons were made across multiple arrays with raw data pulled from different channels for data analysis and comparison to the control data. Data analysis was carried out using Imagene, version 5.1 and Genesight version 4 (Biodiscovery Inc). The mean values from each channel were log2 transformed and normalized using the LOWESS algorithm to remove intensity dependent effects within the calculated values. Normalised values were used to calculate the Cy3/Cy5 fluorescence ratios from experimental and biological repeats (seperate chemostat runs)with all replicates combined. Data was compiled from two aerobic and two anaerobic cultures each with a dye-swap replicate, thus providing two biological repeats within four technical repeats.Presented is the LOG2 normalised data comparing the control (aerobic) with the identified experimental (5, 10, 15, 60 min air depletion) with supplementary material provided in the form of Raw array files.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: ModelOrganism

Release date: 2007-05-10

Last updated: 2007-01-04