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General Information info

Manuscript title Large-scale 13C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast.
PubMed ID 15960801
Journal BMC Genome Biology
Year 2005
Authors Lars M Blank, Lars Kuepfer and Uwe Sauer
Affiliations Institute of Biotechnology, ETH Zürich, 8093 Zürich, Switzerland
Keywords Pentose Phosphate Pathway, Flux Analysis, Glucose Uptake Rate, Oxidative Pentose Phosphate Pathway
Full text article Downloadarticle Blank_2005.pdf
Project name not specified

Experiment Description info

Organism Saccharomyces cerevisiae
Strain CEN.PK113-7D and 38 mutants
Data type flux measurements
Data units mmol/gh
Execution date not specified

Experimental Details info

Temperature (0C) 30
pH 5
Carbon source glucose,
Culture mode batch
Process condition aerobic
Dilution rate (h-1)
Working volume (L) 0.0012
Biomass concentration (g/L) see worksheet
Medium composition

not specified

General protocol information Flux analysis method: 13C constrained MFA

Platform: GC-MS

Methods description - Notes

Glucose, acetate, ethanol and glycerol concentrations in the supernatant were determined with commercial enzymatic kits (Scil Diagnostics, Germany). Organic acids were quantified by high-pressure liquid chromatography (HPLC) using a Supelcogel C8 (4.6 by 250 mm) ion-exclusion column. The column was eluted at 30°C with 2% sulfuric acid at a flow rate of 0.3 ml/min. The organic acids were detected using a PerkinElmer UV detector (Series 2000) at a wavelength of 210 nm. The physiological parameters maximum specific growth rate, biomass yield on glucose, and specific glucose consumption rate were calculated during the exponential growth phase. Analytical procedures and 13C-labeling experiments: All labeling experiments were carried out in batch cultures assuming pseudosteady-state conditions during the exponential growth phase [1, 2]. 13C-labeling of proteinogenic amino acids was achieved either by growth on 5 g/l glucose as a mixture of 80% (w/w) unlabeled and 20% (w/w) uniformly labelled [U-13C]glucose (13C > 99%; Martek Biosciences, Columbia, MD) or 100% [1-13C]glucose (> 99%; Omicron Biochemicals, South Bend, IN). Cells from overnight cultures were harvested by centrifugation and washed using sugar-free MM to remove residual unlabeled carbon sources. Cultures were routinely inoculated to an maximum OD600 of 0.03 and harvested by centrifugation at an OD600 ≤ 1. Residual medium was removed by washing the pellet with water. Cell protein was hydrolyzed for 24 h at 105°C in 6 M HCL and dried in a heating block at 85°C for 6 h. The free amino acids were derivatized at 85°C for 1 h using 15 μl dimethylformamide and 15 μl N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide [3]. Gas chromatography-mass spectrometry (GC-MS) analysis was carried out as reported [2] using a series 8000 GC in combination with an MD800 mass spectrometer (Fisons Instruments, Beverly, MA). --------------------------------------------References---------------------------------------
[1] Fischer E, Sauer U. Eur J Biochem. 2003, 270: 880-891. http://doi.org/dg3q64
[2] Sauer U, Lasko DR, Fiaux J, Hochuli M, Glaser R, Szyperski T, Wüthrich K, Bailey JE. J Bacteriol. 1999, 181: 6679-6688.
[3] Fischer E, Zamboni N, Sauer U. Anal Biochem. 2004, 325: 308-316. http://doi.org/c6kx35

Data file
Downloadfluxes KIMODATAID124_v2.xlsx
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Submission and curation info

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 2018-09-25 13:20:17 UTC

Updated 2018-09-25 13:24:37 UTC

Version 2

Status (reviewed) 2018-09-25 13:23:27 UTC




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