Assays

Maps of cloned genes for rescuing selected clock mutants

E. coli MG1655 and ∆sdhC and ∆frdA isogenic mutant strains were characterized in batch growth curves aerobic and anaerobically. Optical density, glucose consumption and by-product accumulation were measured during growth.

Glucose transporter mutants were analyzed under aerobic and aerobic conditions in batch cultures with glucose as substrate. Acetate formation rates and glucose consumption rates were measured, as well as extracellular cAMP concentrations.

ArcA phosphorylation in chemostat cultures grown at different aerobiosis levels was quantitated by Phos-tag SDS-PAGE gel analysis and subsequent immunodetection of ArcA.

This experiment uses a low-copy plasmid based system (MG1655 Δlac FF(-41.5)/RW50) for measuring FNR activity. Initial acetate calibration of the chemostat with the MG1655 Δlac strain was carried out, with β-galactosidase activity from the FF(-41.5)/RW50 reporter plasmid measured at 100%, 80%, 50%, 20% and 0% aerobiosis levels. Finally, the aerobiosis levels were re-determined by calculating the actual acetate flux in the sampled chemostat runs.

Note: the strain used (MG1655 Δlac) is not the same ...

The task of this assay is to determine the impact of oxygen availability on the concentrations of metabolites from different central metabolic pathways. The focus lies on metabolites connected to glycolysis, tri-carbon-acid-cycle and energy metabolism. All strains have been cultured and analysed according to the SOPs listed below

These files show physiological measurements from the Sheffield Infors chemostat which were made during acetate calibration and also when sampling for the steady-state transcriptional profiles.

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 ...

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).

This assay describes the determination of concentrations and ratio of metabolites of adenine nucleotides (NAD and NADH). These metabolites have been extracted from Escherichia coli MG1655 and isgenic mutant strains.

This assay describes the determination of concentrations and ratio of metabolites of ubiquinones (oxidised and reduced form). These metabolites have been extracted from Escherichia coli MG1655 and isgenic mutant strains.

This .csv file shows the numbers of different cytochrome molecules per cell from steady-state continuously-grown cultures at various aerobiosis levels (0%, 31%, 56%, 85% and 115% AAU).

This assay describes how to analyze gene expression rates via RT-PCR.

This document describes by-product formation rates measured in MG1655 at steady-state conditions in Infors-Multifors-Bioreactors.

The model describes the behaviour of E. coli in a stationary chemostat with different oxygen availability.