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General Information info

Manuscript title Impact of global transcriptional regulation by arcA, arcB, cra, crp, cya, fnr, and mlc on glucose catabolism in Escherichia coli.
PubMed ID 15838044
Journal Journal of Bacteriology
Year 2005
Authors Annik Perrenoud and Uwe Sauer
Affiliations Institute of Biotechnology, ETH, Zurich, Switzerland
Keywords Escherichia coli, aerobic glucose catabolism
Full text article Downloadarticle Perrenoud_2005.pdf
Project name not specified

Experiment Description info

Organism Escherichia coli
Strain BW25113 and mutants
Data type flux measurements
Data units (mmol/g.h)
Execution date not specified

Experimental Details info

Temperature (0C) 37.0
pH not specified
Carbon source glucose,
Culture mode batch
Process condition aerobic
Dilution rate (h-1)
Working volume (L) 0.030-0.050
Biomass concentration (g/L) not specified
Medium composition

M9 medium (per liter deionized water): 0.8 g of NH4Cl, 0.5 g of NaCl, 7.5 g of Na2HPO4 · 2H2O, and 3.0 g of KH2PO4. The following components were sterilized separately and then added (per liter [final volume] of medium): 2 ml of 1 M MgSO4, 1 ml of 0.1 M CaCl2, 0.3 ml of 1 mM filter-sterilized thiamine HCl, and 10 ml of a trace element solution containing (per liter) 1 g of FeCl3 · 6H2O, 0.18 g of ZnSO4 · 7H2O, 0.12 g of CuCl2 · 2H2O, 0.12 g of MnSO4 · H2O, and 0.18 g of CoCl2 · 6H2O. Sterilized glucose was added to a final concentration of 2 or 3 g per liter.

General protocol information Flux analysis method: 13C constrained MFA

Platform: GC-MS

Methods description - Notes

Metabolic flux ratio analysis by GC-MS - Samples for gas chromatography (GC)-mass spectrometry (MS) analysis were prepared as described previously [1]. Briefly, aliquots of 13C-labeled batch cultures were withdrawn during the mid-exponential growth phase, which was defined as an OD600 of 0.8 to 1.2 for aerobic cultures and an OD600 of 0.4 to 0.7 for anaerobic cultures. Cell pellets were hydrolyzed in 6 M HCl at 105 °C for 24 h in sealed microtubes. The hydrolysates were dried under a stream of air at around 60°C and then derivatized at 85°C in 30 µl of dimethylformamide (Fluka, Buchs, Switzerland) and 30µl of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide with 1% (vol/vol) tert-butyldimethylchlorosilane (Fluka) for 60 min. Derivatized amino acids were analyzed with a series 8000 gas chromatograph combined with an MD 800 mass spectrometer (Fisons Instruments, Beverly, Mass.). The GC-MS-derived mass isotope distributions of proteinogenic amino acids were then corrected for naturally occurring isotopes [1]. The corrected mass distributions were related to the in vivo metabolic activities with previously described algebraic equations and statistical data treatment, which quantified nine ratios of fluxes through converging reactions and pathways for the synthesis of five intracellular metabolites [1].
13C-constrained metabolic net flux analysis - Intracellular net carbon fluxes were estimated by using the previously described [2] stoichiometric model.

-----------References------------
[1] Fischer, E., and U. Sauer. 2003. Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. Eur. J. Biochem. 270: 880–891. http://doi.org/dg3q64
[2] Fischer, E., N. Zamboni, and U. Sauer. 2004. High-throughput metabolic flux analysis based on gas chromatography-mass spectrometry derived 13C constraints. Anal. Biochem. 325:308–316. http://doi.org/c6kx35

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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 2013-06-12 15:14:32 UTC

Updated 2014-06-13 09:39:52 UTC

Version 0

Status (reviewed) 2013-12-06 17:17:38 UTC




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