Steady state studies for different oxygen availability in Escherichia coli

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 ...

"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 ...

Goals:

1. Understanding the regulatory principles of Escherichia coli’s electron transport chain (ETC) for varying oxygen conditions in glucose-limited continuous cultures (especially regulatory loops via the transcription factors FNR and ArcA).
2. Explaining the observed phenomena in the measurement data.
3. Predicting unmeasured variables especially of the gene expression regulatory loops.

Means:

1. Experiments (chemostat experiments within the aerobiosis scale).
2. Kinetic modelling (especially ...

Mutant strains which carry deletions of important metabolic enzymes, as well as mutant strains with altered regulation, need to adapt by changing fluxes or gene expression to compensate with the absence/differed concentration of these key enzymes. This may give new insights in the regulation of these pathways/enzymes.

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.

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.

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).

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.

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

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

The file contains simulated data of the electron transport chain model (EcoliETC1) for varying parameter values, i.e. a local sensitivity analysis.

This data file shows results from the different chemostat experiments.Gene expression rates are presented.

The data that was published in Alexeeva et al. [1-3] and Alexeeva [4] is an important prerequisite for SUMO. It is used for the development of the models and for the comparison with the SUMO data. By means of the program EasyNPlot [5] the data in the important plots of [1-3] and of the plot of the ArcA activity in [4] was digitalized. The result can be found in the attached files.

[1] Svetlana Alexeeva, Bart de Kort, Gary Sawers, Klaas J. Hellingwerf, and M. Joost Teixeira de Mattos. Effects of ...

Transcriptional profiles of steady-state chemostat cultures were measured at a range of oxygen levels using microarrays. Oxygen levels were defined by the aerobiosis scale of Alexeeva et al. (2002).

The abscissa of the plots shows the percentage of aerobiosis that is a physiological measure for oxygen availability (http://www.ncbi.nlm.nih.gov/pubmed/11844770).

1. Grey Boxes: Enzymes & Reactions blue lines/symbols: flux in mmol per gramm dry cell weight an hour red lines/symbols: mRNA levels

2. White Boxes: Intracellular and extracellular metabolites blue lines/symbols: concentration of the metabolites (extracellular: mM, intracellular: AU)

3. Yellow Boxes: Aggregated Quantities as yield, ...

This .pdf file shows a graphical representation of the physiological measurements from the Sheffield Infors chemostat which were made during acetate calibration and also when sampling for the steady-state transcriptional profiles.

This .csv file contains growth parameters measured from chemostat runs of the Sheffield Infors chemostats. The runs were carried out for acetate calibration purposes and for sampling for the steady-state transcriptional profiles.

Columns: Date O2_input (% total) Aerobiosis level (%AAU) OD600 Redox (mV) pO2 (μM) DCW (g/L) Viable cell counts (CFU/ml) RespRate (mmol/h/gDCW) RespRate STDEV [Acetate] (mM) Qacet (mmoles/h/gDCW)

A .pdf files showing graphs of FNR activity at varying aerobiosis levels

Top graph shows no acetate re-calibration Bottom graph shows data with acetate re-calibration

This .csv file shows FNR activity at different aerobiosis levels

Columns a_old Desired aerobiosis level (Aerobiosis units or %AAU) Bgal_activity Average FNR reporter activity (Miller units) Bgal_STDEV STDEV of FNR reporter activities (Miller units) rDOT(μM) Dissolved oxygen tension (μM) DCW DCW (g/L) [acetate] Extracellular acetate concentration (mM) Qacetate Acetate flux (mmoles/h/gDCW) a_new Actual aerobiosis level in sampled chemostat

his document contains data for quinone concentrations and ratios of ubichinon and menachinon.These data have been generated for analysis of MG1655 at steady-state conditions in Infors-Multifors-Bioreactors. All measurements have been done in Amsterdam at the group of Prof. Joost Teixeira de Mattos.

Duplicates of samples for quinones were taken after 20 hours of steady state conditions. Chemostat experiments were carried out in Infors-Bioreactors at 0, 20, 50, 80, 100 and 120% aerobiosis. All data ...

This is the csv version of the file: 091016 quinones complete data magdeburg

This document contains data for quinone concentrations and ratios of ubichinon and menachinon.These data have been generated for analysis of MG1655 at steady-state conditions in Infors-Multifors-Bioreactors. All measurements have been done in Amsterdam at the group of Prof. Joost Teixeira de Mattos.

Duplicates of samples for quinones were taken after 20 hours of steady state conditions. Chemostat experiments were carried ...

The model describes the catabolism of Escherichia coli and its regulation. The metabolic reactions are modeled by the thermokinetic model formalism. The model is simplified by assuming rapid equilibrium of many reactions. Regulation is modeled by phenomenological laws describing the activation or repression of enzymes and genes in dependence of metabolic signals. The model is intended to describe the behavior of E. coli in a chemostat culture in depedence on the oxygen supply.

The model is described ...

The model describes the electron transport chain (ETC) of Escherichia coli by ordinary differential equations. Also a simplified growth model based on an abstract reducing potential describing the balance of electron donor (glucose) and electron acceptors is coupled to the ETC. The model should reproduce and predict the regulation of the described system for different oxygen availability within the aerobiosis scale (glucose limited continuous culture<=>chemostat). Therefore oxygen is changed ...

Simplified model of the electron-transport chain(s) (ETC) of Escherichia coli and its regulation by ArcA and FNR. The goal is to demonstrate a hypothetical design principle in the regulatory structure (->partly qualitative parameter values). Oxygen is changed slowly (100% aerobiosis at 1000000 time units) thus the basis variable is not the time but the oxygen flux voxi.

The phosphorylation level of a particular protein can be determined using a procedure based upon western immunoblotting, with Phos-tag™ reagent present in the SDS-PAGE gel. The Phos-tag™ reagent, supplied in the form of Phos-tag™ acrylamide (Wako Pure Chemical Industries, AAL-107), causes proteins to be resolved both on the basis of size and phosphorylation state. This means that phosphorylated and de-phosphorylated forms of the same protein can be distinguished.

This method describes how one can quench metabolism of Escherichia coli and extract metabolites from many kinds of metabolite classes like: nucleotides, sugar-phosphates, organic acids ....

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.

This SOP describes the preparation of E. coli RNA

This file describes how to isolate mRNA from E. coli using the kit from Epicentre, for gene expression analysis via RT-PCR

This document describes the chemostat conditions to run comparable experiments in different bioreactors in different labs.

This SOP describes the SUMO procedure for determining B-galactosidase activities.