Central Carbon Metabolism of Sulfolobus solfataricus (snapshot 1)

An 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 13:52

Contents

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 3PG

Saturation of PGK with 3PG

  • PGK_3PG.xls

PGK kinetics ADP

No description specified

  • PGK_ADP.xls

PGK kinetics ATP

Kinetic characterisation of Phosphoglycerokinase. Measures enzyme reaction rate with increasing concentration of ATP

  • Copy of PGK_ATP.xls

PGK kinetics BPG

No description specified

  • PGK_BPG.xlsx

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

GAPDH kinetics 3PG

No description specified

  • GAPDH_3PG.xlsx

GAPDH kinetics GAP

No description specified

  • GAPDH_GAP.xlsx

GAPDH kinetics NADP

No description specified

  • GAPDH_NADP.xlsx

GAPDH kinetics NADPH

No description specified

  • GAPDH_NADPH.xlsx

GAPDH kinetics Pi

No description specified

  • GAPDH_KPi.xlsx

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

TPI kinetics DHAP

No description specified

  • TIM_DHAP.xlsx

TPI kinetics GAP

No description specified

  • TIM_GAP.xlsx

TPI kinetics PEP inhibition GAP

No description specified

  • TIM_PEP_GAP.xlsx

TPI kinetics PEP inhibition of DHAP

No description specified

  • TIM_PEP_DHAP.xlsx

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.

FBPA/ase kinetics DHAP

No description specified

  • Pase_DHAP.xlsx

FBPA/ase kinetics GAP

No description specified

  • Pase_GAP.xlsx

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.

T degradation DHAP

DHAP concentrations were measured over time at 70C.

  • DHAP.xlsx

T degradation GAP

GAP concentrations were measured over time at 70C.

  • GAP.xlsx

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

T degradation GAP

GAP concentrations were measured over time at 70C.

  • GAP.xlsx

T degradation DHAP

DHAP concentrations were measured over time at 70C.

  • DHAP.xlsx

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

FBPA/ase kinetics DHAP

No description specified

  • Pase_DHAP.xlsx

FBPA/ase kinetics GAP

No description specified

  • Pase_GAP.xlsx

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

FBPAase kinetic model

Mathematical model for FBPAase kinetics

  • FBPald.nb

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 3PG

Saturation of PGK with 3PG

  • PGK_3PG.xls

PGK kinetics ADP

No description specified

  • PGK_ADP.xls

PGK kinetics ATP

Kinetic characterisation of Phosphoglycerokinase. Measures enzyme reaction rate with increasing concentration of ATP

  • Copy of PGK_ATP.xls

PGK kinetics BPG

No description specified

  • PGK_BPG.xlsx

GAPDH modeling

Mathematical model for GAPDH kinetics, saturation of GAP, BPG, NADP, NADPH and Pi.

GAPDH kinetics 3PG

No description specified

  • GAPDH_3PG.xlsx

GAPDH kinetics GAP

No description specified

  • GAPDH_GAP.xlsx

GAPDH kinetics NADP

No description specified

  • GAPDH_NADP.xlsx

GAPDH kinetics NADPH

No description specified

  • GAPDH_NADPH.xlsx

GAPDH kinetics Pi

No description specified

  • GAPDH_KPi.xlsx

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.

TPI kinetics DHAP

No description specified

  • TIM_DHAP.xlsx

TPI kinetics GAP

No description specified

  • TIM_GAP.xlsx

TPI kinetics PEP inhibition GAP

No description specified

  • TIM_PEP_GAP.xlsx

TPI kinetics PEP inhibition of DHAP

No description specified

  • TIM_PEP_DHAP.xlsx

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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Snapshot 1 (2nd Dec 2015)
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