Comment[ArrayExpressAccession] E-GEOD-5084
Public Release Date 2006-08-31
Investigation Title Anaerobic NO-exposed Chemostat Comparison of Wt & norR mutant Responses
Comment[Submitted Name] Anaerobic NO-exposed Chemostat Comparison of Wt & norR mutant Responses
Experiment Description Escherichia coli strains MG1655 and an isogenic norR::Tn5 mutant were 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 0.1 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 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, NOC-5 and NOC-7 were added to the chemostat culture and to the nutrient feed at a final concentration of 10 uM of each. Samples were taken after a period of 5 min exposure to NOC 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 Wild type and norR::Tn5 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 Each strain was grown twice in seperate chemostat runs, exposed to NO. Samples were hybridised as WT vs NorR::Tn5 and each hybridisation had a corresponding dye-swap performed.
Date of Experiment
Term Source Name EFO
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Term Source File http://www.ebi.ac.uk/efo/efo.owl
Person Last Name Poole Pullan Green Poole
Person First Name Robert Steven Jeffrey Robert
Person Mid Initials T R K
Person Email R.poole@shef.ac.uk
Person Affiliation University of Sheffield
Person Phone 01142224447
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Person Address Molecular Biology & Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, South Yorkshire, UK
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Protocol Name P-GSE5084-2 P-GSE5084-1 P-GSE5084-3
Protocol Description ID_REF =
VALUE = test:reference ratio following background correction, log2 transformation, Loess normalisation
CH1_MEAN = Mean data in Cy3 Channel
CH1_BKD_MEAN = Background Mean in Cy3 Channel
CH1_AREA = Spot Area in Cy3 Channel
CH1_BKD_AREA = Background Area in Cy3 Channel
CH1_RAW = Raw signal output data obtained from image analysis algorithm Cy3 Channel
CH1_BKD_RAW = Raw background output data obtained from image analysis algorithm in Cy3 Channel
CH1_SD = Standard Deviation obtained in Cy3 Channel
CH1_BKD_SD = Background Standard Deviation in Cy3 Channel
CH2_MEAN = Mean data in Cy5 Channel
CH2_BKD_MEAN = Background Mean in Cy5 Channel
CH2_AREA = Spot Area in Cy5 Channel
CH2_BKD_AREA = Background Area in Cy5 Channel
CH2_RAW = Raw signal output data obtained from image analysis algorithm in Cy5 Channel
CH2_BKD_RAW = Raw background output data obtained from image analysis algorithm in Cy5 Channel
CH2_SD = Standard Deviation obtained in Cy5 Channel
CH2_BKD_SD = Background Standard Deviation in Cy5 Channel ID_REF =
VALUE = test:reference ratio following background correction, log2 transformation, Loess normalisation
CH1_MEAN = Mean data in Cy3 Channel
CH1_BKD_MEAN = Background Mean in Cy3 Channel
CH1_AREA = Spot Area in Cy3 Channel
CH1_BKD_AREA = Background Area in Cy3 Channel
CH1_RAW = Raw signal output data obtained from image analysis algorithm in Cy3 Channel
CH1_BKD_RAW = Raw background output data obtained from image analysis algorithm in Cy3 Channel
CH1_SD = Standard Deviation obtained in Cy3 Channel
CH1_BKD_SD = Background Standard Deviation in Cy3 Channel
CH2_MEAN = Mean data in Cy5 Channel
CH2_BKD_MEAN = Background Mean in Cy5 Channel
CH2_AREA = Spot Area in Cy5 Channel
CH2_BKD_AREA = Background Area in Cy5 Channel
CH2_RAW = Raw signal output data obtained from image analysis algorithm in Cy5 Channel
CH2_BKD_RAW = Raw background output data obtained from image analysis algorithm in Cy5 Channel
CH2_SD = Standard Deviation obtained in Cy5 Channel
CH2_BKD_SD = Background Standard Deviation in Cy5 Channel background correction, log2 transformation and Loess normalisation were performed using Genesight 4.1 software, copyright BioDiscovery
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Protocol Type bioassay_data_transformation bioassay_data_transformation feature_extraction
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Comment[SecondaryAccession] GSE5084
Comment[GEOLastUpdateDate] 2006-06-18
Comment[AEExperimentType] unknown experiment type
Comment[GEOReleaseDate] 2006-08-30
Comment[ArrayExpressSubmissionDate] 2006-06-14
SDRF File E-GEOD-5084.sdrf.txt