Assay type 'Experimental Assay Type'

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

Measurement of intra- and extra-cellular metabolome.

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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.

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

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

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.

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

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

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

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.

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

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.

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.

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

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

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.

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

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 ...

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

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.

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.

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).

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.

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).

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.

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Submitter: Jay Moore

Assay type: Transcriptomics

Technology type: Custom Array

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

Study: Timeseries 1

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Submitter: Falko Krause

Assay type: Transcriptomics

Technology type: Microarray

Investigation: K+ Starvation in Saccharomyces cerevisiae

Study: Transcriptional Profile

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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.

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.

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 ...

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).

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.

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.

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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.

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

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).

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.

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

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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 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 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 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.

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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.

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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

Submitter: Martijn Bekker

Assay type: Metabolomics

Technology type: Progressive Curve Experiment

Investigation: The Attic

Study: Pre-liminary data from Martijn Bekker

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

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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.

Submitter: Stefan Henrich

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: The Attic

Study: allosteric regulation of pyruvate kinase

extracellular metabolite concentrations measured by 1H-NMR

intracellular metabolite measured by GC/MS and LC/MS

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.

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.

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.

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.

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.

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

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 ...

  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 ...

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

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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 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

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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.

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

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.

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

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.

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.

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.

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

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

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

Concentration of glycolytic intermediates over time

Kinetic characterisation of fructose 1,6-bisphosphate aldolase phosphatase

Temperature degradation of BPG, GAP and DHAP

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

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 ...

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.

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

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

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.

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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.

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Targeted proteomics for peptides related to fatty acid metabolism. Aim: Check which of these proteins we are able to detect in this experiment.

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Here we will use JERM templates with embedded ontologies to describe and annotate example data

Source: Gareth Gillard. (24 June 2016)

TO DO : Library preparation- Tom and Gareth

File types: Raw counts from HTSeq-count - .counts.txt CPM - .CPM.txt log2 scaled CPM - .log2CPM.txt FPKM - .FPKM.txt log2 scaled FPKM - log2FPKM.txt The scaled counts (CPM, FPKM) are derived from the raw counts, and used TMM normalised effective library sizes (using edgeR functions).

For feed switch experiment:

Count tables, separated for liver and gut tissue (can alter sample separation at any point) ...

Record of weight, length and sex of fish sampled after feed switch between vegetable and marine oil, in September 2015 (freshwater) and January 2016 (seawater). Young fry arrived at Solbergstranda 2015-02-05 17:20.

Fatty acids are extracted from samples and converted to fatty acid methyl esters (FAMEs) followed by separation by gas chromatography. This yields fatty acid profiles for each sample as percent of total FAME or milligram FAME per gram of biomass.


Source:

Hei Magny,

Takk!

Et par ting: 1) vi trenger å få orden på data... Lurer derfor på om du kan du lage ett dokument som inneholder alle resultat fra GCen? Uten ...

Fatty acids are extracted from samples and converted to fatty acid methyl esters (FAMEs) followed by separation by gas chromatography. This yields fatty acid profiles for each sample as percent of total FAME or milligram FAME per gram of biomass.


Source:

Hei Magny,

Takk!

Et par ting: 1) vi trenger å få orden på data... Lurer derfor på om du kan du lage ett dokument som inneholder alle resultat fra GCen? Uten ...

Here we will use JERM templates with embedded ontologies to describe and annotate example data 222

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Submitter: Theresa Kouril

Assay type: Experimental Assay Type

Technology type: Enzymatic Activity Measurements

Investigation: 1 hidden item

Study: 1 hidden item

An overview of RNA sequencing data generated in GenoSysFat (and a couple of others).

Source: Email from Simen Rød Sandve to Jon Olav Vik and Fabian Grammes 2017-02-10, titled "RNAseq generert i GSF".

This should be turned into separate RNAseq Assays when we can allocate people for it. Currently the following have records already:

Tissue panel for gene expression in ZF, Med, RT https://fairdomhub.org/assays/324

Tissue panel for gene expression in ZF,Med,RT- RNA sequencing https://fairdomhub.org/assays/395 ...

NB! The files here are the old version for the files in Lipidomics (Experimental Assay)

Lipidomic analysis by LC-MS of tissue samples from the GSF1 feed-switch experiment. Samples were analyzed at NTNU by Zdenka Bartosova and Per Bruheim.

There are two separate data files of lipid analysis in muscle and liver samples. Excel sheets contains both raw and normalised data of compounds abundance. Normalization to all compounds was used as a normalization method.

We have also performed a "normalization ...

Comparison of Kcat values from the model and values from literature.

Submitter: Niels Zondervan

Assay type: Enzymatic Assay

Technology type: Technology Type

Investigation: Modelling of M. pneumoniae metabolism

Study: Core Model training

Protein copy number at 6h, 12h, 24h, 48h, 72h, 96h, average values and SD for the measurements

Submitter: Niels Zondervan

Assay type: Proteomics

Technology type: Technology Type

Investigation: Modelling of M. pneumoniae metabolism

Study: Proteomics analysis

Metabolomics time series measurements for internal metabolites for 6h, 24h and 48h for multiple experiments. Largely based on MAss spectrometry, bioluminescence kits to measure NAD, NADH at 24h, other time points are infered from relative measurements times the absolute measurements at 24h.

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

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RNAseq data for L.ferriphilum samples

Genomics data, for L.ferriphilum Sequencing of the genome and functional annotation

Collection for experimental SOPs

Experimental data for the yeast PGK incubations at 30C, with and without recycling of ATP.

Changes in metabolite concentrations were either quantified via 31P NMR or enzymatically

BPG degradation at 70C

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Transcript profiling by microarray in 4, 6, 8, 12 and 18 h photoperiods, originally published in Flis et al, 2016, Photoperiod-dependent changes in the phase of core clock transcripts and global transcriptional outputs at dawn and dusk in Arabidopsis. doi: 10.1111/pce.12754.

Plant material The same plant material used for transcriptome analysis in (Flis et al., 2016) was the basis of our proteome study. Briefly, Arabidopsis thaliana Col-0 plants were grown on GS 90 soil mixed in a ratio 2:1 (v/v) with vermiculite. Plants were grown for 1 week in a 16 h light (250 μmol m−2 s−1, 20 °C)/8 h dark (6 °C) regime followed by an 8 h light (160 μmol m−2 s−1, 20 °C)/16 h dark (16 °C) regime for one week. Plants were then replanted with five seedlings per pot, transferred for ...

Proteomics data for N15 incorporation into protein in Ostreococcus grown in 12L:12D light:dark cycles.

Quantitative proteomic analysis of Cyanothece ATCC51142 grown in 12L:12D light:dark cycles, using partial metabolic labeling and LC-MS analysis.

Information about the fish from the Kollevåg study

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: Caging study - Kollevåg

Includes physiological informations regarding the fish samples: Weight, length, gender, date sampled, comments regarding physical apperance, in addition to calculations of condition factor (K), hepatosomatic index (HSI) and gonadosomatic index (GSI).

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: PAH/PFAS mixture toxicity in vivo

Data for Figure 2I-2K in Chew et al. PNAS 2014. Experimental conditions: ∼21.3 °C; 12:12-h light/dark cycle; light intensity, 110 μmol·m−2·s−1;mean daytime CO2 level, 375 ppm. The error bars show the SEs of five plants Further detail on the experimental conditions is contained in the public record on the BioDare resource, link to follow

Data for Figures 5D-5F and Supplementary Figure 7B, 7C, including biomass and leaf areas. Image data for leaf areas are included in a .ZIP archive, with two samples as published in 5D. The 'Summary' sheets in the XLSX files include published graphs. Simulation data are included from FMv1. These data were acquired in April 2014, in a separate experiment from the La(er) and Fei-0. Experimental conditions: ∼20.7 °C constant temperature; 12h:12h light/dark cycle; light intensity = 100μmol·m−2·s−1; ...

Data for Figure 4, from the prior publication of Sulpice et al. Mol. Plant 2014: Biomass, net growth and starch levels at end of day and end of night, under light:dark cycles of 4:20, 6:18, 8:16, 12:12 and 18:6 hours.

Data for Figure 3A-3F and Supplementary Figures 2, 3, and 6, including leaf number, biomass and leaf areas. Image data for leaf areas are included in a .ZIP archive. The 'Summary' sheets in the XLSX files often include published graphs. Simulation data are included from FMv1. These data were acquired in June 2012. Experimental conditions: ~22C constant temperature; 12:12-h light/dark cycle; light intensity = 130 μmol·m−2·s−1; average daytime CO2 concentration = 375 ppm. 10 plants per genotype per ...

Data for Figure 3G and Supplementary Figure 4, including gas exchange measurements and photo of the experimental setup. The 'Summary' sheets in the XLSX files often include published graphs. Simulation data are included from FMv1.

These data were acquired in a separate experiment from the biomass, in March 2013. Replication of the earlier biomass study was imperfect, as some plants became a little dry when watering was controlled to reduce moss growth. Sufficient plants grew strongly to measure ...

Enzyme activity of glutathione-s-transferase (Gst) and catalase (Cat) in Kollevåg samples

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Enzymatic Activity Measurements

Investigation: 1 hidden item

Study: Caging study - Kollevåg

Concentrations of steroid hormones E2 and T in female cod plasma from Kollevåg fish

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: Caging study - Kollevåg

Chemical analyses in Kollevåg includes sediment contaminant concentrations, concentrations of various contaminants in cod liver and concentrations of PAH metabolites in cod bile.

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: Caging study - Kollevåg

Western blot results for Kollevåg samples.

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: Caging study - Kollevåg

Analyses of gene expression: qPCR analyses in liver (relative expression) and ovaries (absolute expression).

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: Caging study - Kollevåg

TBARS assay measured oxidative stress as levels of malondialdehyde in samples. Performed in cod liver samples.

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: Caging study - Kollevåg

Biosense Vtg assay measuring concentrations of vitellogenin in cod blood plasma

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: Caging study - Kollevåg

Stranded RNAseq libraries were prepared from 1µg total RNA from liver tissue using TruSeq Stranded mRNA library preparation kit (Illumina, San Diego, USA) using double unique indices (#20022371), according to the manufacturer's instruction (Part 15031057 Rev.E). Libraries were sequenced at the Norwegian Sequencing Centre (NSC). All libraries were pooled, and the same pool was sequenced on 4 flow cell lanes on a HiSeq 3000 machine (Illumina), generating 100bp single-end reads. RNA sequencing files ...

Total lipids are extracted from tissues of white muscle, liver and whole brain from three fish per dietary treatment.

The three different CRISPR target sites within the elovl2 gene (T1-3) were characterised by Sanger sequencing, sequencing on average 8 clones per individual.

RNAseq is utilised to validate the SnpEff annotation predictions for aberrant splicing. For this purpose the percentage exon retention for exons 4, 6 and 7 are calculated using RNAseq data.

Description of observed phenotypic variables in the MOA investigation.

No description specified

Submitter: Ziva Ramsak

Assay type: Q_PCR

Technology type: qPCR

Investigation: MOA - Multiomics analysis of potato response to...

Study: MOA2010-05

No description specified
No description specified

Enzyme activity of the oxidative stress enzymes glutathione S-transferase (Gst) and catalase (Cat) measured in cod liver

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: PAH/PFAS mixture toxicity in vivo

Concentration of the egg yolk precursor protein vitellogenin in cod plasma

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: PAH/PFAS mixture toxicity in vivo

Gene expression of cyp1a and acox1 measured by qPCR in liver of Atlantic cod exposed to PAHs and PFASs

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: PAH/PFAS mixture toxicity in vivo

Semi-quantitative determination of Cyp1a protein amount in cod liver, using the ELISA method.

Submitter: Karina Dale

Assay type: Experimental Assay Type

Technology type: Technology Type

Investigation: 1 hidden item

Study: PAH/PFAS mixture toxicity in vivo