Central Carbon Metabolism of Sulfolobus solfataricus
DownloadAn investigation in the CCM of S. solfataricus with a focus on the unique temperature adaptations and regulation; using a combined modelling and experimental approach.
Created at: 2nd Dec 2015 at 12:52
Carbon loss at high T
No description specified
Reconstituted system reference state
The four purified enzymes were incubated in assay buffer and consumption of 3PG and production of F6P were measured in time, together with GAP and DHAP concentrations.
Metabolite concentrations during reconstituted enzyme incubation
The purified enzymes, PGK, GAPDH, TPI and FBPAase were incubated at 70 C en conversion of 3PG to F6P was followed
- ValidationReference.xlsx
Model simulation and Exp data for reconstituted system
Experimental data for the reconstituted system are plotted together with the model prediction
- combinedPlot.jpg
Model reconstituted system
Mathematical model for the reconstituted system with PGK, GAPDH, TPI and FBPAase.
Metabolite concentrations during reconstituted enzyme incubation
The purified enzymes, PGK, GAPDH, TPI and FBPAase were incubated at 70 C en conversion of 3PG to F6P was followed
- ValidationReference.xlsx
Model simulation and Exp data for reconstituted system
Experimental data for the reconstituted system are plotted together with the model prediction
- combinedPlot.jpg
Mathematical model for the combined four enzyme system
The PGK, GAPDH, TPI and FBPAase were modelled together using the individual rate equations. Closed system.
- ssolfGluconeogenesisOpenAnn.dat
- ssolfGluconeogenesisOpenAnn.xml
- ssolfGluconeogenesisAnn.xml
- ssolfGluconeogenesisClosed.xml
- ssolfGluconeogenesis.xml
- schema-gluconeo.jpg
Model gluconeogenesis
Mathematical model of a subset of reactions comprising the three most temperature sensitive intermediates of the gluconeogenic pathway in S. solfataricus
PGK
Kinetic characterisation of Phosphoglycerokinase
PGK kinetics ATP
Kinetic characterisation of Phosphoglycerokinase. Measures enzyme reaction rate with increasing concentration of ATP
- Copy of PGK_ATP.xls
Model simulation and experimental data for ADP, ATP, 3PG and BPG saturation of PGK
Model simulation and experimental data for ADP, ATP, 3PG and BPG saturation of PGK
- PGK.jpg
Expression of S. solfataricus enzymes in E. coli (SOP_SSO_080913a)
Cloning and heterologous expression of gluconeogenic enzymes in E. coli
- SOP-Cloning and heterologous expression in E.docx
Preparation of cell free extract of recombinant E. coli
Preparation of cell free extracts of the recombinant E. coli strains expressing the respective S. solfataricus enzymes.
- SOP-Preparation of recombinant enzymes.docx
Protein purification from E. coli extracts.
The recombinant enzymes were purified from the cell free extracts of E. coli expressing the respective enzymes.
- SOP-Protein purification.docx
GAPDH
No description specified
Expression of S. solfataricus enzymes in E. coli (SOP_SSO_080913a)
Cloning and heterologous expression of gluconeogenic enzymes in E. coli
- SOP-Cloning and heterologous expression in E.docx
Preparation of cell free extract of recombinant E. coli
Preparation of cell free extracts of the recombinant E. coli strains expressing the respective S. solfataricus enzymes.
- SOP-Preparation of recombinant enzymes.docx
Protein purification from E. coli extracts.
The recombinant enzymes were purified from the cell free extracts of E. coli expressing the respective enzymes.
- SOP-Protein purification.docx
TPI
No description specified
Model simulation and exp data for TIM kinetics, GAP and DHAP saturation, and PEP inhibition.
Model simulation and exp data for TIM kinetics, GAP and DHAP saturation, and PEP inhibition.
- TIM.jpg
Expression of S. solfataricus enzymes in E. coli (SOP_SSO_080913a)
Cloning and heterologous expression of gluconeogenic enzymes in E. coli
- SOP-Cloning and heterologous expression in E.docx
Preparation of cell free extract of recombinant E. coli
Preparation of cell free extracts of the recombinant E. coli strains expressing the respective S. solfataricus enzymes.
- SOP-Preparation of recombinant enzymes.docx
Protein purification from E. coli extracts.
The recombinant enzymes were purified from the cell free extracts of E. coli expressing the respective enzymes.
- SOP-Protein purification.docx
FBPA/ase
Kinetic characterization of the FBPA/ase with respect to GAP en DHAP saturation.
Model simulation and experimental data for DHAP and GAP saturation of FBPA/ase
Model simulation and experimental data for DHAP and GAP saturation of FBPA/ase
- FBPald.jpg
Expression of S. solfataricus enzymes in E. coli (SOP_SSO_080913a)
Cloning and heterologous expression of gluconeogenic enzymes in E. coli
- SOP-Cloning and heterologous expression in E.docx
Preparation of cell free extract of recombinant E. coli
Preparation of cell free extracts of the recombinant E. coli strains expressing the respective S. solfataricus enzymes.
- SOP-Preparation of recombinant enzymes.docx
Protein purification from E. coli extracts.
The recombinant enzymes were purified from the cell free extracts of E. coli expressing the respective enzymes.
- SOP-Protein purification.docx
Experimental assays of the temperature dependent degradation of GAP and DHAP
GAP and DHAP concentrations were measured over time at 70C.
Modelling temperature dependent degradation of GAP and DHAP
The temperature dependent degradation of DHAP and GAP is modelled as an exponential decay process using experimental data for the disintegration at 70C.
Model simulation and Exp data for GAP T deg
A time simulation of the mathematical model is shown together with the experimental data to which the model was fitted.
- TdegGAP.jpg
Model simulation and Exp data for DHAP T deg
A time simulation of the mathematical model is shown together with the experimental data to which the model was fitted.
- TdegDHAP.jpg
GAP T degradation
An exponential decay model was fitted to the experimental data set of GAP degradation at 70C. The model is uploaded as an SBML file. A data file showing the model fit to the experimental data set is included. The rate constant for GAP degradation at 70C was estimated to be 0.056 1/min, equivalent to a half life time of 12.4 min.
- GAPdeg.xml
DHAP T degradation
An exponential decay model was fitted to the experimental data set of DHAP degradation at 70C. The model is uploaded as an SBML file. A data file showing the model fit to the experimental data set is included. The rate constant for DHAP degradation at 70C was estimated to be 0.0225 1/min, equivalent to a half life time of 30.8 min.
- DHAPdeg.xml
FBPAase model
Mathematical model for FBPAase kinetics, saturation of GAP and DHAP
Model simulation and experimental data for DHAP and GAP saturation of FBPA/ase
Model simulation and experimental data for DHAP and GAP saturation of FBPA/ase
- FBPald.jpg
PGK modeling
Mathematical model for PGK kinetics, saturation with ADP, ATP, 3PG and BPG.
Model simulation and experimental data for ADP, ATP, 3PG and BPG saturation of PGK
Model simulation and experimental data for ADP, ATP, 3PG and BPG saturation of PGK
- PGK.jpg
PGK kinetics ATP
Kinetic characterisation of Phosphoglycerokinase. Measures enzyme reaction rate with increasing concentration of ATP
- Copy of PGK_ATP.xls
GAPDH modeling
Mathematical model for GAPDH kinetics, saturation of GAP, BPG, NADP, NADPH and Pi.
GAPDH kinetic model
Mathematical model for GAPDH kinetics, saturation for GAP, BPG, NADP, NADPH, Pi.
- GAPDH.nb
TPI modeling
Mathematical model for TPI kinetics, saturation of GAP and DHAP.
Model simulation and exp data for TIM kinetics, GAP and DHAP saturation, and PEP inhibition.
Model simulation and exp data for TIM kinetics, GAP and DHAP saturation, and PEP inhibition.
- TIM.jpg
TIM kinetic model
Mathematical model for TIM kinetics, GAP and DHAP saturation and PEP inhibition.
- TIM.nb
Gluconeogenic model
Mathematical model for the analysis of carbon loss of BPG, GAP and DHAP in reconstituted system of S. solfataricus at 70C.
Metabolite concentrations during reconstituted enzyme incubation
The purified enzymes, PGK, GAPDH, TPI and FBPAase were incubated at 70 C en conversion of 3PG to F6P was followed
- ValidationReference.xlsx
Mathematical model for the combined four enzyme system
The PGK, GAPDH, TPI and FBPAase were modelled together using the individual rate equations. Closed system.
- ssolfGluconeogenesisOpenAnn.dat
- ssolfGluconeogenesisOpenAnn.xml
- ssolfGluconeogenesisAnn.xml
- ssolfGluconeogenesisClosed.xml
- ssolfGluconeogenesis.xml
- schema-gluconeo.jpg
Pathway optimisation
By varying the input or output flux via PGK and FBPAase manipulation respectively the pathway efficiency can be optimised.
Modelling pathway optimisation
Varying the relative amounts of PGK and FBPAase and analyse the 3PG uptake and F6P production rates.
PGK titration of reconstituted system
Increasing the amount of PGK in the incubation assay and measuring the 3PG and F6P fluxes.
- PGK_titration.xlsx
FBPAase titration of reconstituted system
Increasing the amount of FBPAase in the reconstituted system and measuring the 3PG and F6P fluxes.
- FBPAase_titration.xlsx
Model simulation and experimental data for PGK and FBPAase titrations
Combined plot fo the model simulations and the experimental data for the PGK and FBPAase titrations.
- pgk-ald-titr.jpg
Pathway efficiency for 3PG to F6P conversion as function of PGK or FBPAase concentration
Model simulation showing the effect of varying PGK or FBPAase concentration on the efficiency for 3PG to F6P conversion.
- efficiencyPlot.jpg
Mathematical model for the combined four enzyme system
The PGK, GAPDH, TPI and FBPAase were modelled together using the individual rate equations. Closed system.
- ssolfGluconeogenesisOpenAnn.dat
- ssolfGluconeogenesisOpenAnn.xml
- ssolfGluconeogenesisAnn.xml
- ssolfGluconeogenesisClosed.xml
- ssolfGluconeogenesis.xml
- schema-gluconeo.jpg
PGK and FBPAase titrations
Experimental analysis of pathway fluxes with varying PGK and FBPAase concentrations in the incubation assay.
Model simulation and experimental data for PGK and FBPAase titrations
Combined plot fo the model simulations and the experimental data for the PGK and FBPAase titrations.
- pgk-ald-titr.jpg
PGK titration of reconstituted system
Increasing the amount of PGK in the incubation assay and measuring the 3PG and F6P fluxes.
- PGK_titration.xlsx
FBPAase titration of reconstituted system
Increasing the amount of FBPAase in the reconstituted system and measuring the 3PG and F6P fluxes.
- FBPAase_titration.xlsx
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Created: 2nd Dec 2015 at 12:52
(Since September 2018)
