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General Information info

Manuscript title Nonlinear dependency of intracellular fluxes on growth rate in miniaturized continuous cultures of Escherichia coli.
PubMed ID 16461663
Journal Applied and Environmental Microbiology
Year 2006
Authors Annik Nanchen, Alexander Schicker and Uwe Sauer
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Keywords Escherichia coli, continuous culture, intracellular fluxes
Full text article Downloadarticle Nanchen_2006.pdf
Project name not specified

Experiment Description info

Organism Escherichia coli
Strain MG1655
Data type flux measurements
Data units (mmol/gDW.h)
Execution date not specified

Experimental Details info

Temperature (0C) 37.0
pH 7.0
Carbon source glucose,
Culture mode chemostat
Process condition aerobic
Dilution rate (h-1) several dilution rates
Working volume (L) 0.010
Biomass concentration (g/L) 0.2-0.5
Medium composition

M9 medium contained (per liter of deionized water) 0.8 g NH4Cl, 0.5 g NaCl, 7.5 g Na2HPO4 · 2H2O, and 3.0 g KH2PO4. The following components were sterilized separately and then added (per liter of final volume): 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 ZnSO4 · 7H2O, 0.12 g CuCl2 · 2H2O, 0.12 g MnSO4 · H2O, and 0.18 g CoCl2 · 6H2O. Sterilized glucose was added to a final concentration of 1 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 chromatographymass spectrometry (GC-MS) analysis were prepared as described previously [1]. Briefly, the 13C-labeled chemostat cultures were harvested after the OD600 was stable for at least two volume changes (the minimum continuous operation was seven volume changes). Cell pellets were hydrolyzed in 6MHCl 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 dimethylformamide (Fluka, Switzerland) and 30 µl N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide with 1% (vol/vol) tert-butyldimethylchlorosilane (Fluka, Switzerland) for 60 min [2]. Derivatized amino acids were analyzed on a series 8000 GC combined with an MD 800 mass spectrometer (Fisons Instruments, Beverly, MA). The GC-MSderived 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 obtained with previously described algebraic equations and statistical-data treatment of metabolic-flux ratio analysis [1] using the software Fiat Flux [3].

[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.
[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.
[3] Zamboni, N., E. Fischer, and U. Sauer. 2005. FiatFlux—a software for metabolic flux analysis from 13C-glucose experiments. BMC Bioinformatics 6:209.

Data file
Downloadfluxes KIMODATAID65_v0.xlsx
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Submission and curation info

Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Interests: mathematical modeling, accessible data, use of data

Created 2013-06-12 15:55:08 UTC

Updated 2014-06-13 09:49:13 UTC

Version 0

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

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