This protocol describes the transcriptional profiling of E. coli cultures using microarrays. The protocol utilises RNA isolated as described in another SOP (SUMO RNA isolation from E. coli) and with hybridisation to Ocimum Ocichip E. coli K-12 microarrays.
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Created: 26th Oct 2009 at 14:48
Last updated: 29th Mar 2012 at 21:06
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Version 1 (earliest) Created 26th Oct 2009 at 14:48 by Matthew Rolfe
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Projects: SUMO
Institutions: University of Sheffield
I am a post-doctoral research associate working in Sheffield in the SUMO consortium. My research focuses on transcriptional regulation in E. coli, with particular emaphasis on the transcriptomic analysis of steady-state chemostat cultures using both microarray and qRT-PCR approaches.
Previous experience, especially that gained during my PhD, involved work on Salmonella physiology and lag phase growth, focusing particularly on gene-expression and transcriptional regulation. Other techniques used ...
SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".
The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.
Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...
Projects: BaCell-SysMO, COSMIC, SUMO, KOSMOBAC, SysMO-LAB, PSYSMO, SCaRAB, MOSES, TRANSLUCENT, STREAM, SulfoSys, SysMO DB, SysMO Funders, SilicoTryp, Noisy-Strep
Web page: http://sysmo.net/
"Systems Understanding of Microbial Oxygen responses" (SUMO) investigates how Escherichia coli senses oxygen, or the associated changes in oxidation/reduction balance, via the Fnr and ArcA proteins, how these systems interact with other regulatory systems, and how the redox response of an E. coli population is generated from the responses of single cells. There are five sub-projects to determine system properties and behaviour and three sub-projects to employ different and complementary modelling ...
Programme: SysMO
Public web page: http://www.sysmo.net/index.php?index=55
Organisms: Escherichia coli, Escherichia coli K-12
Changing the oxygen availability leads to an adaptation of Escherichia coli at different biological levels. After pertubation of oxygen in chemostat experiments the microorganism(s) will come back to another steady state. This investigation deals with these stationary responses of Escherichia coli within the aerobiosis scale. The change for different biological variables, in different areas of the organism like the electron transport chain, the TCA cycle or globally is investigated by wildtype ...
Submitter: Michael Ederer
Studies: Basic regulatory principles of Escherichia coli’s electron transport cha..., Determination of the impact of specific enzyme reactions and regulatory ..., Quantitative analysis of catabolic carbon and electron fluxes in E. coli..., The Escherichia coli steady state response to oxygen: from molecular int...
Assays: Analysis of by-product formation rates in MG1655, Analysis of gene expression rates at different aerobiosis levels via RT-PCR, ArcA phosphorylation at different aerobiosis levels (steady states), Characterization of E. coli MG1655 and ∆sdhC and ∆frdA isogenic mutant s..., Determination of intracellular metabolite concentrations, Determination of intracellular redox state by means of NAD/NADH ratio, Determination of intracellular redox state by means of ubiquinones (oxd/..., FNR activity at different aerobiosis levels (steady state), Kinetic modelling of Escherichia coli's electron transport chain, Kinetic modelling of Escherichia coli's electron transport chain coupled..., Literature Data from Alexeeva et al., J. Bacteriol., 2000, 2002, 2003, Measurement of cytochrome numbers, Physiological measurements from Sheffield chemostat, Steady State Oxygen Response of E. coli WT and two Electron Transport Ch..., Transcriptional profiling of steady states at different aerobiosis levels
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Changing the oxygen availability leads to an adaptation of Escherichia coli at different biological levels. After pertubation of oxygen in chemostat experiments there are very quick responses. This investigation deals with this dynamical behaviour (transitions) of Escherichia coli within the aerobiosis scale. The change for different biological variables, in different areas of the organism like the electron transport chain, the TCA cycle or globally is investigated by wildtype and mutants experiments ...
Submitter: Matthew Rolfe
Studies: The Escherichia coli dynamical response to oxygen: from molecular intera...
Assays: Transcriptional profiling of E. coli during anaerobic to aerobic and aer...
Snapshots: No snapshots
Submitter: Matthew Rolfe
Investigation: Dynamical studies for different oxygen availabi...
Assays: Transcriptional profiling of E. coli during anaerobic to aerobic and aer...
Snapshots: No snapshots
Submitter: Michael Ederer
Investigation: Steady state studies for different oxygen avail...
Assays: ArcA phosphorylation at different aerobiosis levels (steady states), Determination of intracellular redox state by means of NAD/NADH ratio, Determination of intracellular redox state by means of ubiquinones (oxd/..., FNR activity at different aerobiosis levels (steady state), Literature Data from Alexeeva et al., J. Bacteriol., 2000, 2002, 2003, Measurement of cytochrome numbers, Physiological measurements from Sheffield chemostat, Steady State Oxygen Response of E. coli WT and two Electron Transport Ch..., Transcriptional profiling of steady states at different aerobiosis levels
Snapshots: No snapshots
This assay involved the determination of transcriptional profiles at 0, 2, 5, 10, 15 and 20 minutes through aerobic to anaerobic gas transitions and anaerobic to aerobic gas transitions. In each case an aerobic or anaerobic steady state was created, RNA sampled (0 min) and then the gas supply changed. RNA samples were then taken from the time at which the gas supply was changed.
For anaerobic conditions 5% CO2, 95% N2 was used.
The full transcriptional dataset is available from ArrayExpress ...
Submitter: Matthew Rolfe
Assay type: Transcriptional Profiling
Technology type: Microarray
Investigation: Dynamical studies for different oxygen availabi...
Organisms: Escherichia coli : K-12/MG1655 (wild-type / wild-type)
SOPs: Evans Medium, SUMO RNA isolation from E. coli, SUMO Transcriptional profiling of E. coli, SUMO chemostat conditions
Data files: Dissolved oxygen tension aerobic to anaerobic .pdf, Dissolved oxygen tension anaerobic to aerobic .pdf, Entire transcriptional dataset - aerobic to ana..., Entire transcriptional dataset - anaerobic to a..., Filtered dataset - anaerobic to aerobic .csv, Filtered datset - aerobic to anaerobic transiti...
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The transcriptional profiles of steady state E. coli cultures at a range of aerobiosis levels were determined. Two biological replicates and two technical replicates were carried out. Microarrays were carried out in a reference style (i.e. RNA vs a gDNA reference).
Submitter: Matthew Rolfe
Assay type: Transcriptomics
Technology type: Microarray
Investigation: Steady state studies for different oxygen avail...
Organisms: Escherichia coli : K-12/MG1655 (wild-type / wild-type)
SOPs: SUMO RNA isolation from E. coli, SUMO Transcriptional profiling of E. coli
Data files: Steady state transcriptional profiles at differ...
Snapshots: No snapshots