DataEntryID 118 General information Manuscript title: Metabolic and Transcriptional Response to Cofactor Perturbations in Escherichia coli. PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/20299454 Journal: Journal of Biological Chemistry Year: 2010 Authors: Anders K. Holm, Lars M. Blank, Marco Oldiges, Andreas Schmid, Christian Solem, Peter R. Jensen and Goutham N. Vemuri Affiliations: Department of Systems Biology, Center for Systems Microbiology, Technical University of Denmark, 2800 Denmark Keywords: F1Fo ATPase, Glycolysis, Metabolic Regulation, Transcription Regulation, Tricarboxylic Acid (TCA) Cycle, Cofactor Perturbation, Escherichia coli, NADH Oxidase, Soluble ATPase, Systems Biology Full text article: https://kimosys.org/rails/active_storage/blobs/eyJfcmFpbHMiOnsibWVzc2FnZSI6IkJBaHBBdUVFIiwiZXhwIjpudWxsLCJwdXIiOiJibG9iX2lkIn19--125c85d305b840a45d360e219645e9744e504e1c/Holm_2010.pdf Project name: not specified Experiment description Organism: Escherichia coli Strain: MG1655 and 2 over-expression strains (NOX, ATPase) Data type: flux measurements Data units: mmol/gdw h Execution date: not specified Experimental details Temperature (°C): 37 pH: not specified Carbon source: glucose Culture mode: batch Process condition: aerobic Dilution rate (h⁻¹): — Working volume: 0.1 L Biomass concentration (g/L): Biomass yield from glucose (g dry cell weight/g glucose): WT = 0.43 ± 0.02, NOX = 0.29 ± 0.03 and ATPase = 0.22 ± 0.00 Medium composition: Defined MOPS medium [1] supplemented with 0.1% glucose and 200 μg/ml erythromycin. The flasks were agitated at 150 rpm to ensure aerobic conditions. General protocol information: Type analysis list: flux ratio; Platform list: GC-MS, LC-MS; Methods description: Biomass samples for determining the fluxes were centrifuged (5000 × g, 10 min at 4 °C) and harvested in 150 μl of 6 m HCl and hydrolyzed at 105 °C for 24 h before drying at 85 °C. The hydrolyzate was dissolved in 50 μl of dimethyl formamide and derivatized using 30 μl of N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide. The mixture was incubated at 85 °C with shaking at 550 rpm for 60 min (Orbital Shaker CSA, Thermo Scientific) before GC-MS analysis, using previously described protocols [2,3]. From the mass isotope distribution patterns of the proteinogenic amino acids, relative pathway contributions (metabolic flux ratios) were computed that were used as constraints to infer metabolic activity [2,3]. An aliquot of 5 ml of the culture from mid-exponential phase was quenched in hot phenol (80 °C) and subsequently frozen at −20 °C for measuring the concentration of intracellular metabolites. The samples were processed as described previously [4] and analyzed using LC-MS/MS for quantitative determination of central carbon metabolites and cofactors. The values were normalized with biomass concentrations at the time of sampling. --------------------------------------------References--------------------------------------- [1] Neidhardt F. C., Bloch P. L., Smith D. F. (1974) J. Bacteriol. 119, 736–747. [2] Fischer E., Sauer U. (2003) Eur. J. Biochem. 270, 880–891. http://doi.org/dg3q64 [3] Fischer E., Zamboni N., Sauer U. (2004) Anal. Biochem. 325, 308–316. http://doi.org/c6kx35 [4] Luo B., Groenke K., Takors R., Wandrey C., Oldiges M. (2007) J. Chromatogr. A. 1147, 153–164. http://doi.org/cxdj7w Data file: http://kimosys.org/repository/118/download?parameter=1248; Alternative formats: no files uploaded Submission and curation Entered by: Administrator KiMoSys Created: 2018-08-24 18:15:24 UTC Updated: 2020-04-24 16:10:37 UTC Version: 1 Status: (reviewed) 2018-08-24 18:15:40 UTC Views: 179 Downloads: 58