Assays

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.

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.

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.

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.

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

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

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.

Measurement of intra- and extra-cellular metabolome.

test

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

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

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.