Assays

Dear SEEK users, this Assay is just an example Excel sheet for intracellular metabolites concentration measurements performed using cell culture growing in chemostat

Concentration of glycolytic intermediates over time

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

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

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.

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, ...

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.

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

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.

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

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

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.

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.

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

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

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.

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.

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.

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.

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.

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

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.

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.