DataEntryID 35
    General information
      Manuscript title: Multiple High-Throughput Analyses Monitor the Response of E. coli to Perturbations.
      PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/17379776
      Journal: Science
      Year: 2007
      Authors: Nobuyoshi Ishii, Kenji Nakahigashi, Tomoya Baba, Martin Robert, Tomoyoshi Soga, Akio Kanai, Takashi Hirasawa, Miki Naba, Kenta Hirai, Aminul Hoque, Pei Yee Ho, Yuji Kakazu, Kaori Sugawara, Saori Igarashi, Satoshi Harada, Takeshi Masuda, Naoyuki Sugiyama, Takashi Togashi, Miki Hasegawa, Yuki Takai, Katsuyuki Yugi, Kazuharu Arakawa, Nayuta Iwata, Yoshihiro Toya, Yoichi Nakayama, Takaaki Nishioka, Kazuyuki Shimizu, Hirotada Mori, Masaru Tomita
      Affiliations: Institute for Advanced Biosciences, Keio University
      Keywords: Escherichia coli, perturbations response, chemostat, single knockouts
      Full text article: no file uploaded
      Project name: not specified

    Experiment description
      Organism: Escherichia coli
      Strain: WT K-12 BW25113 and mutants
      Data type: flux measurements
      Data units: (mmol/gDW/h)
      Execution date: not specified

    Experimental details
      Temperature (°C): 37.0
      pH: 7.0
      Carbon source: glucose
      Culture mode: chemostat
      Process condition: aerobic
      Dilution rate (h⁻¹): 0.1, 0.2, 0.4, 0.5 and 0.7
      Working volume: 1.0 L
      Biomass concentration (g/L): See Supplementary files of the original article (Tabel S3).
      Medium composition: Synthetic medium: 48mM Na2HPO4, 22mM KH2PO4, 10mM NaCl, 45mM (NH4)2SO4, 4g/L glucose, 1mM MgSO4, 1mg/L thiamin.HCL,  5.6 mg/L CaCl2, 8 mg/L FeCl3, 1 mg/L MnCl2.4H2O, 1.7 mg/L ZnCl2, 0.43 mg/L CuCl2.2H2O, 0.6 mg/L CoCl2.2H2O and 0.6 mg/L Na2MoO4.2H2O
      General protocol information: Type analysis list: 13C constrained MFA; Platform list: GC-MS;
      Methods description: 13C-labeling experiment - for metabolic flux analysis, 13C-labeling experiments were initiated after taking samples for transcriptome, proteome and metabolome analysis. For experiments with gene disruptants, the feed medium containing 4 g/l of natural glucose was replaced by an identical medium containing 0.4 g/l of [1-13C] glucose, 0.4 g/l of uniformly labeled [U-13C] glucose and 3.2 g/l of natural glucose. For experiments examining the effect of changes in dilution rate on flux distributions, the composition of glucose in the feed medium was changed to 0.8 g/l of [1-13C] glucose, 0.8 g/l of [U-13C] glucose and 2.4 g/l of natural glucose. After two residence times, for gas chromatography-mass spectrometry (GC-MS) analysis, E. coli cells were harvested by centrifugation.
GC-MS analysis - the cells obtained from about 250 ml of culture were suspended in 4 ml of 6 M HCl and then hydrolyzed at 105 ºC for 16 h. After cooling, HCl was evaporated with a centrifugal evaporator (CVE-3100, Tokyo Rikakikai Co., Ltd., Japan). The dried hydrolysate was resuspended in water and then filtrated through a 0.22-μm pore size filter (Millipore Co., USA). The filtrate was dried again and redissolved in 1.5 ml of acetonitrile. For derivatization, the resulting 80 μl of biomass hydrolysate dissolved in acetonitrile was mixed with an equal volume of N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide and then incubated at 110 ºC for 30 min. After cooling, the derivatized sample was used for the GC-MS analysis using a TurboMass Gold mass spectrometer (Perkin Elmer, USA). In the present study, two fragment ions, [M-57]+ and [M-159]+, of tert-butyldimethylsilylated (TBDMS-) amino acids (Ala, Gly, Val, Ile, Pro, Ser, Met, Phe, Asp, Glu and Tyr) were monitored. The analytical conditions for GC-MS were as described by Zhao et al [1].
Estimation of metabolic flux distribution - for metabolic flux analysis, we constructed a basic stoichiometric reaction model for the main metabolic pathways including glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, the glyoxylate shunt and the anaplerotic pathway. The biomass content reported by Li et al. [2] was used for calculations.

--------------------References---------------
[1] J. Zhao, K. Shimizu, J. Biotechnol. 101, 101 (2003). http://doi.org/cdjhrn
[2] M. Li, P. Y. Ho, S. Yao, K. Shimizu, J. Biotechnol. 122, 254 (2006). http://doi.org/cr7qpd 
      Data file: http://kimosys.org/repository/35/download?parameter=1125; 
      Alternative formats: no files uploaded

    Submission and curation
      Entered by: Administrator KiMoSys
      Created: 2013-04-22 11:42:01 UTC
      Updated: 2020-04-24 16:10:28 UTC
      Version: 3
      Status: (reviewed) 2013-12-06 17:17:38 UTC
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