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Series GSE2129 Query DataSets for GSE2129
Status Public on Apr 06, 2005
Title Transcriptional responses of Anaerobically grown Escherichia coli to GSNO under defined chemostat conditions.
Platform organism Escherichia coli K-12
Sample organism Escherichia coli
Experiment type Expression profiling by array
Summary Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in defined media containing 54 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 1 litre, and the dilution rate 0.2 h-1. In order to establish anaerobic growth, nitrogen was sparged through the chemostat medium prior to inoculation and throughout the course of the experiment at a rate of 0.2 l/min. No dissolved oxygen was detectable using the OxyProbe. Sodium fumarate was added to anaerobic medium at a final concentration of 50 mM to act as a terminal electron acceptor. Cells were grown as above to steady-state, At steady-state, GSNO was added to the chemostat culture and to the nutrient feed at a final concentration of 200 uM unless otherwise stated. Samples were taken immediately prior to the addition of GSNO and after a period of 5 min exposure to GSNO for subsequent analysis using microarrays. Cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers.
Equal quantities of RNA from control and GSNO-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values.

Keywords: dose response
 
 
Contributor(s) Flately J, Barrett J, Pullan ST, Hughes MN, Green J, Poole RK
Citation(s) 15647275
Submission date Jan 06, 2005
Last update date Mar 16, 2012
Contact name Jason Barrett
E-mail(s) J.A.Barrett@sheffield.ac.uk
Phone (+44) 114 2222834
Organization name University of Sheffield
Department Department of Molecular Biology and Biotechnology
Street address Firth Court, Western Bank
City Sheffield
State/province South Yorkshire
ZIP/Postal code S10 2TN
Country United Kingdom
 
Platforms (1)
GPL534 MWG E. coli K12 Array
Samples (4)
GSM38546 Anaerobic GSNO Chemostat Run 1
GSM38547 Anaerobic GSNO Chemostat Run 1 Dye Swap
GSM38548 Anaerobic GSNO Chemostat Run 2
Relations
BioProject PRJNA91743

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