Metabolic and Transcriptional Response to Cofactor Perturbations in Escherichia coli. http://www.ncbi.nlm.nih.gov/pubmed/20299454 Journal of Biological Chemistry 2010 Anders K. Holm, Lars M. Blank, Marco Oldiges, Andreas Schmid, Christian Solem, Peter R. Jensen and Goutham N. Vemuri Department of Systems Biology, Center for Systems Microbiology, Technical University of Denmark, 2800 Denmark F1Fo ATPase, Glycolysis, Metabolic Regulation, Transcription Regulation, Tricarboxylic Acid (TCA) Cycle, Cofactor Perturbation, Escherichia coli, NADH Oxidase, Soluble ATPase, Systems Biology https://kimosys.org/rails/active_storage/blobs/eyJfcmFpbHMiOnsibWVzc2FnZSI6IkJBaHBBdU1FIiwiZXhwIjpudWxsLCJwdXIiOiJibG9iX2lkIn19--8f68435eb7e4ac85d6ca1cacd3a9a802e0d93ce3/Holm_2010.pdf not specified Escherichia coli MG1655 and 2 over-expression strains (NOX, ATPase) metabolites at steady-state μmol/g DCW not specified 37 °C not specified glucose batch aerobic — h⁻¹ 0.1 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 g/L 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. Sampling Method: aliquot of 5 ml of the culture from mid-exponential phase; Quenching: quenched in hot phenol (80 °C) and subsequently frozen at −20 °C.; Extraction list: methanol-water; Analysis list: LC-MS; 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 http://kimosys.org/repository/119/download?parameter=1250; Administrator KiMoSys 2018-08-25 15:22:03 UTC 2020-04-24 16:10:37 UTC 0 (reviewed) 2018-08-25 15:24:09 UTC 298 77