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General Information info

Manuscript title Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis.
PubMed ID 19917605
Journal The Journal of Biological Chemistry
Year 2010
Authors Roelco J. Kleijn, Joerg M. Buescher, Ludovic Le Chat, Matthieu Jules, Stephane Aymerich and Uwe Sauer
Affiliations Institute of Molecular System Biology, ETH Zurich, CH-8093 Zurich, Switzerland
Keywords Metabolism, Metabolism/Gluconeogenesis, Metabolism/Intermediary, Metabolism/Regulation, Metabolism/Tricarboxylic Acid Cycle, Methods/Mass Spectrometry, flux analysis, metabolomics
Full text article Downloadarticle Kleijn_2010.pdf
Project name not specified

Experiment Description info

Organism Bacillus subtilis
Strain Wild-type BSB168 trp+
Data type flux measurements
Data units not specified
Execution date not specified

Experimental Details info

Temperature (0C) 37
pH not specified
Carbon source glucose, glucose + malate, malate
Culture mode batch
Process condition aerobic
Dilution rate (h-1) -
Working volume (L) 0.03
Biomass concentration (g/L) dry weight was inferred from a predetermined conversion factor of 0.48 g of cells/A600
Medium composition

M9 minimal medium (per liter): 8.5 g of Na2HPO4.2H20, 3 g of KH2PO4, 1 g of NH4Cl, 0.5 g of NaCl. The following components were sterilized separately and then added (per liter of final medium): 1 ml of 0.1 m CaCl2.2H2O, 1 ml of 1 m MgSO4.7H2O, 1 ml of 50 mm FeCl3.6H2O, and 10 ml of trace salt solution. The trace salts solution contained (per liter): 170 mg of ZnCl2, 100 mg of MnCl2.4H2O, 60.0 mg of CoCl2.6H2O, 60.0 mg of Na2MoO4.2H2O, and 43.0 mg CuCl2.2H2O. When preparing the medium, the base salts were added first followed by CalCl2, MgSO4, FeCl3, and finally the trace elements.

General protocol information Flux analysis method: 13C constrained MFA

Platform: GC-MS

Methods description - Notes

Physiological Parameters: Extracellular substrate and byproduct concentrations were measured by HPLC analysis using an Agilent 1100 series HPLC stack in combination with an Aminex HPX-87H polymer column (Bio-Rad). Sugars were detected with a refractive index detector, and organic acids were detected with a UV-visible detector. Specific rates were calculated by regression analysis for at least five time points during the exponential growth phase as described previously [1]. Cell growth was monitored photometrically at 600 nm, and cell dry weight was inferred from a predetermined conversion factor of 0.48 g of cells/A600 [2]. All physiological parameters were determined during the exponential growth phase, typically ranging from 0.10 to 1.5 A600. Metabolic Flux Analysis: Metabolic fluxes were derived using whole isotopologue analysis [3,4]. In short, the procedure uses the cumomer balances and cumomer to isotopologue mapping matrices introduced by Wiechert et al. [5] to calculate the isotopologue distributions of metabolites in a pre-defined stoichiometric network model for a given flux-set. The flux-set that gives the best correspondence between the measured and simulated 13C-label distribution is determined by non-linear optimization and denoted as the optimal flux-fit. All calculations were performed in Matlab 7.6.0.
----------------------------------------------References-------------------------------------------
[1] Sauer U., Lasko D. R., Fiaux J., Hochuli M., Glaser R., Szyperski T., Wüthrich K., Bailey J. E. (1999) J. Bacteriol. 181, 6679–6688. [2] Tännler S., Decasper S., Sauer U. (2008) Microb. Cell Fact. 7, 19. http://doi.org/cjbk3x [3] van Winden W. A., van Dam J. C., Ras C., Kleijn R. J., Vinke J. L., van Gulik W. M., Heijnen J. J. (2005) FEMS Yeast Res. 5, 559–568. http://doi.org/cgg3tw [4] Kleijn R. J., van Winden W. A., van Gulik W. M., Heijnen J. J. (2005) FEBS J. 272, 4970–4982. http://doi.org/bxpccx [5] Wiechert W, Möllney M, Isermann N, Wurzel M, de Graaf AA (1999). Biotechnol Bioeng 66: 69–85.

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Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Homepage: http://kdbio.inesc-id.pt/kimosys
Interests: mathematical modeling, accessible data, use of data

Created 2018-07-11 22:06:12 UTC

Updated 2018-07-11 22:34:25 UTC

Version 4

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