Assays

240 Assays visible to you, out of a total of 446

- Comparison of metabolic flux distribution in carbon core metabolism (EMP, PPP, TCA) of Bacillus subtilis under 3 different conditions: "salt-free" reference, "stress" chemostat, "osmoprotected" chemostat.
- Model created using OpenFLUX and Microsoft Excel
- Model computed using MatLAB

2D Gels in wild type and mutants grown or incubated under several conditions: starvation, potassium limiting or different growth phases.

Is it possible to see changes in the proteome after starvation in 2D- Gels? Preparation of 2D Gels of cells incubated different periods of time in the absence of potassium.

Contributor: Falko Krause

Assay type: Experimental Assay

Technology type: Technology type

Investigation: K+ Starvation in Saccharomyces cerevisiae

Study: Proteomic Studies

No description specified
No description specified

Absolute quantification of proteins using heavy labeled QconCAT as an internal standard and quantifying the native proteins in the complex sample via scheduled Multiple Reaction Monitoring(MRM) .

Further fluxes (ammonium, chloride, calcium) will be measured dependent on the capacities of the MIFE/FLISE technique.

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.

MG1655 and mutant strains with defects in glucose transport systems were analyzed in aerobic and anaerobic batch cultures.

Some examples of transcriptomics templates for Affymetrix data that conform to the MAGE-TAB specification. These templates were taken from the GEO website (http://www.ncbi.nlm.nih.gov/geo/info/spreadsheet.html) and modified to conform to the SysMO-JERM (Just enough Results Model) for transcriptomics.
Using these templates will mean easier submission to GEO/ArrayExpress and greater consistency of data in SEEK.

Kinetic characterisation en mathematical modelling of ALD.

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

Based on Hess et al. (2006) ammonium is suspected to be transported via Trk1,2 under potassium shortage. The ammonium concentration in the medium will be determined for several time points under the conditions of Navarrete et al. (2010).

Contributor: Falko Krause

Assay type: Experimental Assay

Technology type: Technology type

Investigation: K+ Starvation in Saccharomyces cerevisiae

Study: Ion Flux Changes

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

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

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

No description specified
No description specified

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

Theoretical analysis of hypothetical sigma factor competition.
Based on the model 'transcription factor competition' possible dynamics of sigma factor competition are simulated and analysed using Lineweaver-Burk representations.

Theoretical analysis of hypothetical sigma factor competition.
Based on the model 'transcription factor competition' possible dynamics of sigma factor competition are simulated and analysed using Lineweaver-Burk representations.

For analyzing the binding of CcpA-HPrSer46P-complexes to various cre-elements, Surface Plasmon Resonance was used. All operations were carried out on a Biacore X instrument (Biacore, Uppsala, Sweden). Biotinylated cre DNA was coupled on a Neutravidin coated chip in flowcell two, a biotinylated reference DNA in flowcell one. For visualizing only the interactions of the CcpA-HPrSer46P-complex with the cre elements, CcpA was saturated with 50µM HPrSer46P. Titrations were carried out with 5-100nM
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S. pyogenes M49 (591), E. faecalis V583, and L. lacis NZ9000 and their isogenic ldh deletion mutants were grown glucose free CDM-LAB medium in BIOLOG phenotype microarray plates PM01 and PM02. With this assay the abilitiy of the strains to grow on 190 different carbon sources was determined in 96 well format.

Biomass weight during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

No description specified

Contributor: Ron Henkel

Biological problem addressed: Cell cycle

Investigation: Hands-on: Model Management in SEEK

Study: Hands-On: Tyson1991 - Cell Cycle 6 var

A boolean network was created using booleannet (after experimenting with Squad and CellNetAnalyzer). This network can be simulated and visualized using additional software components that will be part of the pyMantis CMS that is developed by the Translucent project.

The main input is the ENA review paper (Function and Regulation of the Saccharomyces cerevisiae ENA Sodium ATPase System, Ruiz&Ariño 2007) and the papers referenced.
Another source are the papers linked from the ENA page of SGD http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=ENA1

Cellular size and granularity (measured by FACS) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

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.

Enzymes involved in butanediol formation from pyruvate in L. Lactis and E. faecalis were characterized with respect to their Km's for their substrates and their Vmaxes

Contributor: Martijn Bekker

Assay type: Experimental Assay

Technology type: Initial rate experiment

Investigation: The Attic

Study: Pre-liminary data from Martijn Bekker

S. pyogenes wildetype, an arcA- and a glnA-deletion mutants were grown in CDM-LAB cultures at pH 6.5 and 7.5 and at a growth rate of 0.05

Mutant strains which lack one or more of the glucose transport systems were analyzed in aerobic chemostat cultures and compared to batch cultures.

This Excel template is an example taken from the GEO web site (http://www.ncbi.nlm.nih.gov/geo/info/spreadsheet.html#GAtemplates) which has been modified to conform to the SysMO JERM (Just Enough Results Model).
Using templates helps with searching and comparing data as well as making it easier to submit data to public repositories for publications.

Simulation of double mutants and perturbations and time series samples using for Sample 1 only OE mutants of which we update the enzyme concentrations. For each second mutant the enzyme concentrations in case of OE and KO mutants in updated and the metabolite concentrations of the second sample are loaded in the model.
Using this approach the model approximately predicts combinatorial effects of OE mutations with other mutations, perturbations and time series concentrations.

Contributor: Niels Zondervan

Biological problem addressed: Modelling Analysis

Investigation: 1 hidden item

Study: Core model combinatorial predictions

We will compare two different procedures to extract ATP from yeast cells: Standard kit procedure (hot Tris/EDTA) and Serrano procedure (cold perchloric acid). In addition we have tested different condition as it turned out that some are important.

Samples obtained form the central fermentation facility of Sulfosys have been compared using iTRAQ (isobaric tag for relative and absolute quantification). A pilot experiment resulted in creation of SOP and initial data on cells grown at 70 and 80C

Investigation of all steady state pH-values between pH 5.7 and 4.5 (pH 5.5, 5.3, 5.1, 4.9, 4.7).

Comparison of the transcriptome at steady state in acidogenesis and at steady state in solventogenesis.

Investigation of the dynamic switch at pH values between 5.8 and 4.5 (pH 5.5, 5.3, 5.1, 4.9, 4.7 and 4.5).

The pilot experiment was conducted in order to create SOPs and to gain insight in transcriptome of cells grown at 70 and 80C

In order to construct an in vivo-like buffer for S. pyogenes, the intracellular concentrations of Fe, K, Mg, Mn Na, P and S elements were determined via ICP-AES (inductively coupled plasma atomic emissionspectroscopy) method at the Institute of Land Use, University of Rostock. The samples for the analysis were obtained from a steady state culture grown on CDM-LAB with glucose.

We here create a kinetic model for a single enzyme within the T. brucei trypanothione synthesis pathway, the enzyme trypanothione synthetase based on the insights from the laboratory experiments

No description specified

The current-voltage relation for Trk1,2 will be determined according to the conditions of Kuroda et al.

The current-voltage relation for Trk1,2 will be determined for various external potassium concentrations.

No description specified

Current- voltage relations for will be determined for various internal potassium concentrations.

Proton fluxes ensue a change in the membrane potential to which the potassium uptake responds. The membrane potential changes depend on the extrusion of protons, buffering capacities of the media and experimental parametes.

Contributor: Falko Krause

Biological problem addressed: Modelling Analysis

Investigation: TRK1,2 Transport Systems of Saccharomyces cerev...

Study: Kinetic properties of Trk

Mutants with a linear respiratory chain consisting of NADH Dehydrogenase II and one of the terminal oxidases cytochrom bo, cytochrome bd I or cytochrome bd II were growth in chemostats with defined oxygen supply. The amounts of biomass formed and of acetate and formate produced were determined.

Determination of essential amino acids for Streptococcus pyogenes

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

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.

Analytical methods and computational analyses (regression, fitting) will be employed to find properties of the Trk system under different external conditions.

Contributor: Falko Krause

Biological problem addressed: Modelling Analysis

Investigation: TRK1,2 Transport Systems of Saccharomyces cerev...

Study: Kinetic properties of Trk

Mutants with linear respiratory chains were grown under SUMO chemostat conditions at different defined aerobiosis levels. The ArcA phoshorylation state as determined.

Dynamics of extracellular metabolites (glc, pyr, suc, lac, gly, ac, etoh, fum, mal, cit, including loss of akg, g3p, 2pg, 3pg, r5p, f6p, g6p, 6pg) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

Dynamics of intracellular metabolites (pyr, suc, fum, mal, akg, pep, g3p, 2pg, 3pg, cit, r5p, f6p, g6p, 6pg, ATP, ADP, AMP, UTP, GTP, inosine, NAD+, IMP, UDP, NADP+, CTP, AdenyloSuccinate, NADPH, trehalose) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines,
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Dynamics of macromolecules (total RNA) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

Kinetic characterisation en mathematical modelling of ENO.

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

Based on initial cell material, series of SOPs connected with assays of enzymes involved in Central Carbon Metabolism of S. solfataricus have been developed.

Experimental data for the conversion of 3PG to F6P and the gluconeogenic pathway intermediates

External pH changes for the conditions of Navarrete et al. (2010) will be estimated from the internal pH changes and the volume ratio of cell sample to medium.

External concentration changes under the conditions of Navarrete et al. (2010) will be estimated from the internal concentration changes and the volume ratio of cell sample to medium.

extracellular metabolite concentrations measured by 1H-NMR

Mathematical model for FBPAase kinetics, saturation with DHAP and GAP

In order to get uniform data from all geographically separated partners in a consortium, central fermentation facility has been set-up. Based on empirical data, standard procedures for growing S. solfataricus cells have been developed.

The multi-compartmental metabolic network of Arabidopsis thaliana was reconstructed and optimized in order to explain growth stoichiometry of the plant both in light and in dark conditions. Balances and turnover of energy (ATP/ADP) and redox (NAD(P)H/NAD(P)) metabolites as well as proton in different compartments were estimated. The model showed that in light conditions, the plastid ATP balance depended on the relationship between fluxes through photorespiration and photosynthesis including both
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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
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Kinetic characterisation en mathematical modelling of G3PDH.

No description specified

Kinetic characterisation en mathematical modelling of GAPDH.

Mathematical model for GAPDH kinetics, saturation with BPG, NADPH, NADP, GAP and Pi

Contributor: Jacky Snoep

Biological problem addressed: Enzymology

Investigation: Central Carbon Metabolism of Sulfolobus solfata...

Study: Model Gluconeogenesis

Mutant strains with linear electron transport chain were grown in chemostat cultures at different defined aerobiosis levels. Expression of selected genes was determined by Real Time RT-PCR

Some generic examples of transcriptomics templates that conform to the MAGE-TAB specification. These templates were created and modified from templates produced by ArrayExpress and GEO.
These templates are generic and non-specific for any particular array platform.

No description specified

This assay is for method development to quantify intra- and extra-cellular metabolites on T. brucei 427 bloodstream form using isotope ratio based MS technique with 13C-labelled E. coli extract

Metabolic network of Enterococcus faecalis including primary metabolism, polysaccharide metabolism, purine and pyrimidine biosoynthesis, teichoic acid biosynthesis, fatty acid and phospholipid bioynthesis, amino acid metabolism, vitamins and cofactors

Metabolic network of S. pyogenes including primary metabolism, polysaccharide metabolism, purine and pyrimidine biosoynthesis, teichoic acid biosynthesis, fatty acid and phospholipid bioynthesis, amino acid metabolism, vitamins and cofactors

No description specified

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

Global sensitivity analysis of a kinetic model to determine the sensitivities for each parameter, over a wide parameter range. We used the elementary effects method.

S. pyogenes was grown in rich medium, strongly concentrated and glucose-pulsed in a MES buffer. Intracellular metabolite concentration is followed in time.

No description specified

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

Cytoscape based analysis and yED based representation of clostridial Reactomes

Kinetic characterisation en mathematical modelling of HK.

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

How does internal potassium changes (decreases) during several hours of potassium starvation. Is there a limit for internal potassium decrease? Sampling potassium content in cells during starvation
The relative membrane potential will be measured according to the conditions of Navarrete et al. (2010). Various mutants will be tested for their effect.
To estimate the relative changes of plasma-membrane potential, the diSC3(3) fluorescent probe was used and the changes in cell fluorescence monitored
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Determination of protein content at several times.

The potassium content measurements of Navarrete et al. (2010) are the basis for potassium content analysis in various mutants (Nha1, Trk1, Trk2).

No description specified

Using PCA, three components, beam size 8.
Clustering via MCL from Biolayout Express 3D

Data is taken from "Genome-Wide Gene Expression Analysis of the Switch between Acidogenesis and Solventogenesis in Continuous Cultures of Clostridium acetobutylicum." Grimmler et al. 2011
DOI: 10.1159/000320973

Development of bioinformatic tools to investigate the role of transcription factors and 14-3-3 proteins in the regulation of genes involved in cation homeostasis.

Contributor: Falko Krause

Biological problem addressed: Modelling Analysis

Investigation: TRK1,2 Transport Systems of Saccharomyces cerev...

Study: Bioinformatic studies

No description specified
No description specified

Contributor: Falko Krause

Biological problem addressed: Modelling Analysis

Investigation: TRK1,2 Transport Systems of Saccharomyces cerev...

Study: Promotor Anaysis

No description specified

Contributor: Sebastian Curth

Biological problem addressed: Modelling Analysis

Investigation: Modular Model Building

Study: Automated Model Building

Elucidation of protein networks involved in the regulation of cation homeostasis using protein interaction datasets.

Contributor: Falko Krause

Biological problem addressed: Modelling Analysis

Investigation: TRK1,2 Transport Systems of Saccharomyces cerev...

Study: Bioinformatic studies

To estimate the differnces of internal pH between the wild type, single trk1 and trk2 or double trk1 trk2 mutants, the pH-dependent variant of GFP pHluorin was expressed in cells from a multicopy plasmid, cells were grown under various conditions, and the cell fluorescence was monitored.

26 intracellular metabolites (amino acids, polyamines, TCA intermediates) in T. brucei exposed to methylene blue have been absolutely quantified using isotope ratio based MS technique.

26 intracellular metabolites (amino acids, polyamines, TCA intermediates) in T. brucei under pH stress (pH8.7) have been absolutely quantified using isotope ratio based MS technique.

intracellular metabolite measured by GC/MS and LC/MS

Pilot experiment concerning metabolome of S. solfataricus was conducted in order to acquire SOPs regarding the technique and gain insight on differences in metabolite concentrations at 70 and 80C

We use BSA115 strain which lacks RsbU and RsbW proteins. Therefore, there is limited post-transcriptional regulation of sigmaB activity.
SigmaB itself is placed downstream of Pspac, inducible by IPTG. The lacZ reporter gene is downstream of Pctc promoter.
IPTG concentrations of 0.1, 0.2 and 1 mM are added in mid-exponential phase at an OD of appr. 0.3. The whole experiment runs for about eight hours.

We use BSA115 strain which lacks RsbU and RsbW proteins. Therefore, there is limited post-transcriptional regulation of sigmaB activity.

There occurs an unexpected drop in the beta-Gal activity after sigB induction. This modelling effort aims to clarify the reasons.

Using Taverna for mining and MATLAB for conversion into specific formats (cytoscape, SBTOOLBOX2)

In this experiment we glucose-pulsed an E. faecalis cultures re-suspended in 100 mM MES buffer at pH 6.5. Samples were taken in time to study intra- and extracellular metabolites. These data are used to construct a kinetic model of the catqabolism of E. faecalis

In this experiment we glucose-pulsed an L. lactiss cultures re-suspended in 100 mM MES buffer at pH 6.5. Samples were taken in time to study intra- and extracellular metabolites. These data are used to construct a kinetic model of the catabolism of E. L. lactis

No description specified

Measurements on Km, Vmax and allosteric activation or inhibition of the main L-lactate dehydrogenase

Measurements on Km, Vmax and allosteric activation or inhibition of the heterologously expressed (E. coli) and purifiied main L-lactate dehydrogenase

Artificial transcription elongation compexes are assembled in vitro using synthetic deoxy-oligonucleotides (representing template and non template DNA strands), radiolabelled RNA (representing nascent transcript) and purified RNA polymerase. After high salt wash the incorrect NTP is added and reaction is allowed to proceed for the various amounts of time. Reaction is stopped by addition of formamide-containing loading solution and the products are resolved on high-percentage denaturing polyacryamide
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No description specified

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

No description specified

Extracellular metabolites in T. brucei at different stage of cell growth have been quantified absolutely by isotope ratio based MS technique using uniformly 13C-labelled E. coli extract. This is the case study for method development of absolute quantification for metabolic flux analysis.

Intracellular metabolites in T. brucei at different stage of cell growth have been quantified absolutely by isotope ratio based MS technique using uniformly 13C-labelled E. coli extract. This is the case study for method development of absolute quantification for metabolic flux analysis.

Kinetic characterisation and mathematical modelling of LDH.

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

No description specified

Km values of pyruvate kinase of different organisms without/with allosteric effector molecules collected from literature.

Contributor: Stefan Henrich

Assay type: Experimental Assay

Technology type: Technology type

Investigation: The Attic

Study: allosteric regulation of pyruvate kinase

1. Preparation of B. subtilis cultures

Inoculate cells from -80°C stocks in 10 ml time-lapse microscopy (TLM) medium (62 mM K2HPO4 , 44mM KH2PO4, 15 mM (NH4)2SO4, 6.5 mM sodium citrate, 0.8 mM MgSO4, 0.02 % casamino acids, 27.8 mM glucose, 0.1 mM L-tryptophan, the pH was set to 7 using a KOH solution) supplemented with antibiotics, if necessary.
Grow the cells overnight in a shake flask (30°C, 225 rpm).
The following morning, dilute the cells 1:10 in pre-warmed chemically defined medium (CDM) (62
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No description specified

L. lactis, E. faecalis and S. pyogenes are referred to as LAB because of the fact that in the presence of glucose lactate is produced as the main fermentation product . This metabolic pathway is relatively inefficient, since only 2 ATP are generated from one glucose molecule. All three LAB possess the genetic make up for mixed acid fermentation, a more effective way of fermentation generating 3 ATP per molecule of glucose. All three genomes reveal (at least) two genes encoding a lactate dehydrogenase
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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).

To estimate the relative level of plasma-membrane potential in the wild type, single trk1 and trk2 or double trk1 trk2 mutants, the diSC3(3) fluorescent probe was added to cells grown under various conditions and the levels of cell fluorescence were monitored.

To estimate the relative level of plasma-membrane potential in the wild type, single trk1 and trk2 or double trk1 trk2 mutants, the diSC3(3) fluorescent probe was added to cells grown under various conditions and the levels of cell fluorescence were monitored.

Genome scale metabolic model of Sulfolobus solfataricus
specific scenario: modelling of L-fucose degradation pathways

No description specified

Metabolite profiling on T. brucei exposed to methylene blue has been carried out using LC-MS to investigate metabolic changes caused by oxidative stress

Measurement of intra- and extra-cellular metabolome.

Measurements of external metabolites based on growth curve data.
Flux estimates for uptake of external metabolites such as glucose and production rates for external metabolites lactate and acetate

Contributor: Niels Zondervan

Assay type: Experimental Assay

Technology type: Technology type

Investigation: 1 hidden item

Study: Metabolomics measurements

This Excel template is the general (master) template for any type of metabolomics data. It can be used as it is, or extended and modified to create a more specific templates for particular technologies and assay types.

Lumped kinetic model of L. lactis glycolysis, formulated with ordinary differential equations. Simulations are in line with experimental data

Modelling the degradation of BPG, GAP and DHAP at high temperature

Based on a kinetic model a description of the potassium current is achieved. Its properties with respect to changes in membrane potential and potassium concentrations are derived.

Contributor: Falko Krause

Biological problem addressed: Modelling Analysis

Investigation: TRK1,2 Transport Systems of Saccharomyces cerev...

Study: Kinetic properties of Trk

The RNAseq data on mRNA processing and mRNA decay were used to update a previously published model and to interrogate which process should be dependent on mRNA length

The dynamic model describes response of yeast metabolic network on metabolic perturbation (i.e. glucose-pulse). One compartmental ODE-based model of yeast anaerobic metabolism includes: glycolysis, pentose phosphate reactions, purine de novo synthesis pathway, purine salvage reactions, redox reactions and biomass growth. The model describes metabolic perturbation of steady state growing cells in chemostat.

This assay is designed to measure the decay kinetics of mRNA in T. brucei blood forms. T. brucei lacks the canonical transcriptional regulation employed by other eukaryotes through transcription factors, and relies almost entirely on regulation of mRNA decay and further downstream steps in order to control gene expression.
3 replicates of 8 time points were taken to measure mRNA abundance in the cell using RNA-seq. The first time point was fot WT, untreated cells; the second was 5 min after the
...

Some examples of transcriptomics templates for NimbleGen data that conform to the MAGE-TAB specification. These templates were taken from the GEO website (http://www.ncbi.nlm.nih.gov/geo/info/spreadsheet.html) and modified to conform to the SysMO-JERM (Just enough Results Model) for transcriptomics.
Using templates will mean easier submission to GEO/ArrayExpress upon publication and greater consistency of data in SEEK for easier searching and comparing.

No description specified
No description specified

Contributor: Theresa Kouril

Assay type: Experimental Assay

Technology type: Enzymatic activity measurements

Investigation: 1 hidden item

Study: 1 hidden item

OptKnock algorithm was used as part of a study in RobOKoD: microbial strain design for (over)production of target compounds. (http://fairdomhub.org/publications/236). It was used to generate a strain of e.coli for producing butanol, that was then compared to an experimental strain.

Cells were grown to mid-exponential phase (OD600nm ~0.2) in GM17 medium at 37°C (with 0.15 mM ZnSO4 where relevant) and 84 ml of culture was mixed by inverting with 8.4 ml of fixing solution (50 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 30% (v/v) formaldehyde) and incubated at room temperature for 30 min. Cells were disrupted and crosslinked DNA was sheared by sonication. Antibodies coupled to magnetic beads were used to pull down cross-linked complexes. DNA was purified, amplified,
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Kinetic characterisation en mathematical modelling of PFK.

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

Kinetic characterisation en mathematical modelling of PGI.

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

Kinetic characterisation en mathematical modelling of PGK.

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

Mathematical model for PGK kinetics, saturation with ADP, ATP, 3PG and BPG.

Contributor: Jacky Snoep

Biological problem addressed: Enzymology

Investigation: Central Carbon Metabolism of Sulfolobus solfata...

Study: Model Gluconeogenesis

Kinetic characterisation en mathematical modelling of PGM.

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

L. lactis, S. pyogenes and E. faecalis were grown in C-limited chemostat cultures at various pH's and dilution rates. General flux distribution, yields and other physiological factors were studied.

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.

Kinetic characterisation en mathematical modelling of PK.

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

The external potassium changes will be monitored by the MIFE and FLISE technique. This allows an estimation of internal potassium changes by determining an initial concentration.

Is internal potassium affected by mutations in the Trk1,2 system? Under which conditions? Potassium measurements in wild type and TRK mutants grown and/or incubated under several external conditions.

The potassium fluxes will be estimated from the internal and external concentration changes.

How potassium starvation regulates the parameters of rubidium (potassium) transport. Analysis of transport characteristics during the starvation process. Kinetic characteristics of rubidium transport.

Contributor: Falko Krause

Assay type: Experimental Assay

Technology type: Technology type

Investigation: K+ Starvation in Saccharomyces cerevisiae

Study: Ion Flux Changes

The potassium influx after the addition of a certain amount of KCl to a potassium free medium, followed by the injection of glucose will be measured by using the MIFE and FLISE technique. This reveals a time course of potassium. Also the external potassium concentrations will be measured.

Genes are transcribed in polysictronic messages (pre-mRNA) that are destined for either maturation into mRNAs, or degradation. Since transcription regulation is non-existent with few exceptions, the rate of pre-mRNA processing, together with mRNA decay and translation rates, are believed to control gene expression. In this assay, 2T1 blood form trypanosomes are subject to treatment by ActinomycinD for 5 minutes, inhibiting transcription. The cells are harvested, depleted for ribosomal RNA, and
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No description specified

Is protein content of trk1,2 mutants affected? Determination of proteins in wild type and TRK mutants

What are the main proteins identified? Spots sampling and identification by MS

Contributor: Falko Krause

Assay type: Experimental Assay

Technology type: Technology type

Investigation: K+ Starvation in Saccharomyces cerevisiae

Study: Proteomic Studies

No description specified

Examples of proteomics templates for gele electrophoresis data that conform to the MIAPE-GE specification

Some examples of proteomics templates for Mass Spectrometry data that conform to the MIAPE specification

The external pH changes will be monitored by the MIFE and FLISE technique. This allows an estimation of internal pH changes by determining an initial pH. pH changes will be also determined by using green fluorescent protein dyes. Relating the proton efflux and the change of internal pH allows an estimate of the proton buffering capacity.

Related to the internal pH changes the proton efflux will be estimated from the internal and external concentration changes.

Contributor: Falko Krause

Assay type: Experimental Assay

Technology type: Technology type

Investigation: K+ Starvation in Saccharomyces cerevisiae

Study: Ion Flux Changes

In parallel with the potassium influx the efflux of protons is monitored by measuring the external proton concentration changes with MIFE or FLISE.

No description specified

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

Pyruvate formate-lyase (PFL) is an important enzyme in the metabolic pathway of lactic acid bacteria (LAB) and is held responsible for the regulation of the shift between homolactic acid to mixed acid fermentation. PFL catalysis the reversible reaction of acetyl-CoA and formate into pyruvate and CoA. A glycyl radical, who is regenerated within the reaction, is involved; therefore, PFL works only under strictly anaerobic conditions. For its activation, the C-terminal domain has to bind to the
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Contributor: Stefan Henrich

Biological problem addressed: Modelling Analysis

Investigation: The Attic

Study: Pyruvate formate-lyase (PFL)

In vitro reconstitution of the PGK, GAPHD, TPI and FBPAase enzymes from S. solfataricus

Despite high similarity in sequence and catalytic properties, the L-lactate dehydrogenases (LDH) in lactic acid bacteria (LAB) display differences in their regulation which may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose-1,6-bisphosphate (FBP), phosphate (Pi) and ionic strength (NaCl concentration) on 6 LDHs from 4 LABs studied at pH 6 and pH 7. We find: (1) The extent of activation by FBP (Kact)
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S.pneumoniae D39 cells (wild type and delta greA) were grown in C+Y medium and cells were harvested for total RNA isolation at mid-exponential growth (OD600nm 0.3 for wt, 0.25 for delta greA). Total RNA was isolated as described before (Kloosterman et al 2006).
The total RNA samples were examined by capillary electrophoresis.
dephosphorylated with antarctic phosphatase followed by treatment with polynucleotide kinase (PNK).
Afterwards, samples were poly(A)-tailed using poly(A) polymerase. Then a
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RobOKoD algorithm was, designed then implemented as part of a study in RobOKoD: microbial strain design for (over)production of target compounds. (http://fairdomhub.org/publications/236). It was used to generate a strain of e.coli for producing butanol, that was then compared to an experimental strain. It was shown to perform better than similar methods (OptKnock, and RobustKnock).

RobustKnock algorithm was used as part of a study in RobOKoD: microbial strain design for (over)production of target compounds. (http://fairdomhub.org/publications/236). It was used to generate a strain of e.coli for producing butanol, that was then compared to an experimental strain.

No description specified

A example of an RT-PCR Excel template. RT-PCR is Reverse Transcriptase PCR (NOT to be confused with Real Time PCR, which is normally referred to as qPCR)

This template was taken from the GEO website (http://www.ncbi.nlm.nih.gov/geo/info/spreadsheet.html) and modified to conform to the SysMO-JERM (Just enough Results Model) for transcriptomics.
Using templates will mean easier submission to public databases on publication and greater consistency of data in SEEK.

The membrane potential of cells grown overnight in a certain concentration of KCl will be determined, to reveal a relation between external KCl and a possibly stable membrane potential. See also "2D-Gel Electrophoresis " Assay for further details.

The pH of cells grown overnight in a certain concentration of KCl will be determined, to reveal a functional relationship between external KCl and a stable pH. See also "2D-Gel Electrophoresis" Assay for further details.

The potassium content of cells grown overnight in a certain amount (0.1, 0.2, 0.5, 1, 5, 20, 50, 200 mM) of KCl will be determined. Additionally the potassium content of cells grown overnight in high potassium and shifted to the amounts of potassium used in the former experiment. After growing overnight again in the lower potassium, the cells should contain finally a comparable potassium concentration than the cells grown in the respecting KCl in the first experiment.

The volume of cells grown overnight in a certain concentration of KCl will be determined, to reveal a relation between the osmotic effects of external KCl and the cell volume. See also 1.4.1. for further details.

No description specified

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model validation

Steady state concentrations of extracellular metabolites in yeast Saccharomyces cerevisiae in anaerobic chemostat at D = 0.1 h-1 on minimal medium

experimentally measured extracellular fluxes in yeast Saccharomyces cerevisiae in anaerobic glucose limited chemostat (D=0.1 h-1) on minimal medium

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

Steady state study of the effect of altering gene regulation on yields of end-products, focusing on butanol.

The stressosome is an important sensor of environmental stresses in B. subtilis. It is formed by three protein types that form an icosahedral geometric protein complex. There are uncertanties how protein interactions take place, what the effects on the response behaviour of activation and inhibition of phosphorylation among proteins is, and what kind of proximal signal activates the stressosome in the first place.
To answer these questions a computational modelling approach was developed. This
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Measurements of acetone, butanol, acetate, butyrate and ethanol taken during dynamic shift (pH 5.8, 5.5, 5.3, 5.1, 4.9, 4.7, 4.5) and at steady state (pH 5.7, 5.5, 5.3, 5.1, 4.9, 4.7, 4.5).

No description specified

Temperature degradation of BPG, GAP and DHAP

test

No description specified

We developed a new metabolomics protocol, which involved a comparison of different harvesting techniques, quenching solutions and extraction methods. Cell leakage and metabolite recovery was determined by ATP measurements

Using TFinfer2 to analyse data from "Characterization of MG1655 and mutant strains under conditions of glucose excess and limitation"

B. subtilis was grown in SMM media with glucose as carbon source and the samples for RNA were harvested OD578nm- 1.0). The stress conditions that were applied over here are growthat 57°C, 16°C, 1.2M Nacl and 37°C(control).
All the samples were analysed for transcriptome as biological triplicates.

B. subtilis was grown in M9 media with glucose as carbon source and the samples were harvested during exponential phase (OD600nm- 0.4), early stationary phase(OD600nm- 1.3), late stationary phase(OD600nm- 1.0).
All the samples were analysed for transcriptome as biological triplicates.

No description specified

Contributor: Falko Krause

Assay type: Transcriptomics

Technology type: Microarray

Investigation: K+ Starvation in Saccharomyces cerevisiae

Study: Transcriptional Profile

Time courses of the internal pH changes under the conditions of Navarrete et al. (2010) will be obtained. The usage of different mutants (transport systems and regulation factors) will reveal the influence and key systems of pH regulation.

To estimate the changes of internal pH, the pH-dependent variant of GFP pHluorin is expressed in cells from a multicopy plasmid, and the changes in cell fluorescence are monitored during 5 hours of incubation in YNB-F growth media without added potassium.

Time-dependent simulations of the dynamic switch between acidogenesis and solventogenesis based on the metabolic network and pH-dependent regulation of the enzymes.

Kinetic characterisation en mathematical modelling of TPI.

Contributor: Dawie Van Niekerk

Biological problem addressed: Modelling Analysis

Investigation: Glucose metabolism in Plasmodium falciparum tro...

Study: Model construction

Mathematical model for TPI kinetics, saturation with GAP and DHAP, and inhibition by 3PG and PEP

Contributor: Jacky Snoep

Biological problem addressed: Enzymology

Investigation: Central Carbon Metabolism of Sulfolobus solfata...

Study: Model Gluconeogenesis

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
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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).

Transcriptome analysis for the samples harvested from Chemostat cultivated samples.

No description specified
No description specified

Contributor: Jay Moore

Assay type: Transcriptomics

Technology type: Custom array

Investigation: Metabolism of Streptomyces coelicolor (SysMO ST...

Study: Timeseries 1

Assay for the isolation of intact Plasmodium falciparum trophozoites from infected red blood cells and the preparation of a cell free extract that can be used for kinetic analyses.

This assay is designed to obtain the in vitro kinetic data of T. brucei recombinant trypanothione synthetase. The enzyme catalyzes the ATP-dependent ligation of spermidine (Spd) and GSH to generate glutathionylspermidine (Gsp) and also of Gsp and GSH to finally produce trypanothione (T(SH)2). The data was obtained in an spectrophotometric assay that links ADP production with NADH consumption through the piruvte kinase and lactate dehydrogenase.

TbTryS activity was measured at 37°C in the in vivo-like buffer. All substrate stock solutions were prepared in the in vivo-like buffer and the pH was adjusted to 7.0. The assays were performed in a final volume of 2 ml and contained 0.2 mM NADH, 1 mM phosphoenolpyruvate, 4 units pyruvate kinase, 4 units L-lactate dehydrogenase, 0.17 µM TbTryS, 2.1 mM ATP and varying amounts of glutathione, and spermidine.

What is the relationship between the activity of Trk1,2 system and cell volume? Is cell volume affected by lack of the main potassium uptake systems? Comparison of cell volume in wild type and TRK mutants.

Is cell volume affected by potassium starvation?

The volume reduction as a response to the shift of cells from a medium of high potassium to potassium free medium will be studied in mutants lacking certain membrane transporters like Trk1,2 Nha1, etc. The conditions for the experiments follow Navarrete et al. (2010). Additionally knockouts of related regulation proteins (SAP155, SAP185) will be tested. For each mutant several time points will be measured to generate time courses.

A interaction network analysis tool (currently based on the BioGrid - PSICQUIC web services) was created that helps to discover interactions of Yeast proteins. The tool will at some point be freely available on the www as part of the pyMantis CMS created within the Translucent project.

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