Assays

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.

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

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 ...

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.

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

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).

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

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).

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).

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, ...

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 ...

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 ...

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.

Transcriptome analysis for the samples harvested from Chemostat cultivated samples.

Determination of essential amino acids for Streptococcus pyogenes

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) .

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 ...

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

extracellular metabolite concentrations measured by 1H-NMR