|
| Status |
Public on Sep 01, 2006 |
| Title |
Anaerobic NO-exposed Chemostat Comparison of Wt & norR mutant Responses |
| Platform organism |
Escherichia coli K-12 |
| Sample organism |
Escherichia coli |
| Experiment type |
Expression profiling by array
|
| Summary |
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
Keywords: Mutant Comparison, stress response, nitric oxide, NorR, chemostat, continuous culture
|
| |
|
| Overall design |
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.
|
| |
|
| Contributor(s) |
Pullan ST, Green JR, Poole RK |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| Submission date |
Jun 15, 2006 |
| Last update date |
Sep 13, 2013 |
| Contact name |
Robert Poole |
| E-mail(s) |
R.poole@shef.ac.uk
|
| Phone |
01142224447
|
| Organization name |
University of Sheffield
|
| Department |
Molecular Biology & Biotechnology
|
| Lab |
F13
|
| Street address |
Firth Court, Western Bank
|
| City |
Sheffield |
| State/province |
South Yorkshire |
| ZIP/Postal code |
S10 2TN |
| Country |
United Kingdom |
| |
|
| Platforms (1) |
|
| Samples (4)
|
| GSM114612 |
Anaerobic NO exposed Chemostat Comparison of Wt & norR mutant, Run 1 |
| GSM114613 |
Anaerobic NO exposed Chemostat Comparison of Wt & norR mutant, Run 1 dye swap |
| GSM114614 |
Anaerobic NO exposed Chemostat Comparison of Wt & norR mutant, Run 2 |
| GSM114615 |
Anaerobic NO exposed Chemostat Comparison of Wt & norR mutant, Run 2 dye swap |
|
| This SubSeries is part of SuperSeries: |
| GSE5098 |
Aerobic and anaerobic transcriptional responses of wild type, hmp and norR to strains to NO in a chemostat |
|
| Relations |
| BioProject |
PRJNA104509 |
Less...
More...