Matthew Rolfe

"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 ...

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 ...

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).

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 document shows the ArcA phosphorylation levels at different aerobiosis units, measured using Phos-tag gels, western immunoblotting. Exposed films were quantitated using ImageJ and the results shown here.

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).

This .csv file contains the filtered datset of the aerobic to anaerobic transition. Values are shown if they show a statistically significant change relative to the 0 minute transcriptional profile (t-test p<0.05 and 2-fold cut-off).

This .csv file contains the filtered datset of the anaerobic to aerobic transition. Values are shown if they show a statistically significant change relative to the 0 minute transcriptional profile (t-test p<0.05 and 2-fold cut-off).

This is a pdf showing a graph of the dissolved oxygen tension of the culture during an aerobic to anaerobic transition.

This is a pdf showing a graph of the dissolved oxygen tension of the culture during an anaerobic to aerobic transition.

This .csv file contains the entire transcriptional dataset of the aerobic to anaerobic transition. The values shown are the gene-expression ratio at 2, 5, 10, 15 and 20 minutes relative to the 0 minute timepoint.

This .csv file contains the entire transcriptional dataset of the anaerobic to aerobic transition. The values shown are the gene-expression ratio at 2, 5, 10, 15 and 20 minutes relative to the 0 minute timepoint.

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

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.

The Gene-doctoring method of lambda-red deletion (Lee et al., 2009) was modified slightly to create chromosomal mutations of fnr.

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