DataEntryID 124 General information Manuscript title: Large-scale 13C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast. PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/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: https://kimosys.org/rails/active_storage/blobs/eyJfcmFpbHMiOnsibWVzc2FnZSI6IkJBaHBBdTBFIiwiZXhwIjpudWxsLCJwdXIiOiJibG9iX2lkIn19--29c7615b86322c0b0e0b5ad9dc69de5b18deea8f/Blank_2005.pdf Project name: not specified Experiment description Organism: Saccharomyces cerevisiae Strain: CEN.PK113-7D and 38 mutants Data type: flux measurements Data units: mmol/gh Execution date: not specified Experimental details Temperature (°C): 30 pH: 5 Carbon source: glucose Culture mode: batch Process condition: aerobic Dilution rate (h⁻¹): — Working volume: 0.0012 L Biomass concentration (g/L): see worksheet Medium composition: not specified General protocol information: Type analysis list: 13C constrained MFA; Platform list: GC-MS; Methods description: 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: http://kimosys.org/repository/124/download?parameter=1260; Alternative formats: no files uploaded Submission and curation Entered by: Administrator KiMoSys Created: 2018-09-25 13:20:17 UTC Updated: 2020-04-24 16:10:37 UTC Version: 2 Status: (reviewed) 2018-09-25 13:23:27 UTC Views: 193 Downloads: 48