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General Information info

Manuscript title Latent Pathway Activation and Increased Pathway Capacity Enable Escherichia coli Adaptation to Loss of Key Metabolic Enzymes.
PubMed ID 16319065
Journal Journal of Biological Chemistry
Year 2006
Authors Stephen S. Fong, Annik Nanchen, Bernhard O. Palsson and Uwe Sauer
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich CH-8093, Switzerland
Keywords Escherichia coli, latent pathway activation
Full text article Downloadarticle Fong_2006.pdf
Project name not specified

Experiment Description info

Organism Escherichia coli
Strain MG1655 and (pgi, ppc, pta, tpi) mutants
Data type flux measurements
Data units mmol/gdw h
Execution date not specified

Experimental Details info

Temperature (0C) 37
pH not specified
Carbon source glucose,
Culture mode batch
Process condition aerobic
Dilution rate (h-1)
Working volume (L) 0.03
Biomass concentration (g/L) see spreadsheet
Medium composition

M9 minimal medium supplemented with 2 g/liter of glucose in 500-ml Erlenmeyer flasks. M9 medium contained (per liter of 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. At the start of evolution, initial precultures of each mutant were grown overnight in LB medium before being transferred to minimal medium for adaptive evolution.

General protocol information Flux analysis method: flux ratio, 13C constrained MFA

Platform: GC-MS

Methods description - Notes

After 8 h of incubation at 37 °C and constant shaking, LB precultures were used to inoculate M9 medium precultures that were grown overnight for inoculation of cultures for physiological or 13C-labeling experiments. For 13C-labeling experiments, glucose was added either entirely as the 1-13C-labeled isotope isomer (>99%; Euriso-top, GIF-sur-Yvette, France) or as a mixture of 20% (w/w) U-13C (>98%; Isotech, Miamisburg, OH) and 80% (w/w) natural glucose. Cell growth was monitored by following the A600. Glucose and acetate concentrations were determined enzymatically using commercial kits (Beckman-Coulter (Zurich, Switzerland) or Dispolab (Dielsdorf, Switzerland)). Other organic acids in culture supernatants were detected by high pressure liquid chromatography analysis (PerkinElmer Life Sciences) at a wavelength of 210 nm, using a Supelcogel C8 column (4.6 × 250 mm) at 30 °C and a mobile phase of 2% (v/v) sulfuric acid at a flow rate of 0.3 ml/min.
The following physiological parameters were determined during the exponential growth phase as described previously [1]: maximum growth rate, biomass yield on glucose, specific glucose consumption rate, and specific byproduct production rates, using a predetermined correlation factor of 0.44 g of cellular dry weight per liter and A600 unit.
Metabolic Flux Ratio (METAFoR) Analysis by Gas Chromatography-Mass Spectrometry—Samples for gas chromatography-mass spectrometry analysis were prepared as described previously [2]. 13C-Constrained Net Flux Analysis—Intracellular net fluxes were estimated with a stoichiometric model that contained all major pathways of central carbon metabolism [3]. See more information in the original article.
-----------------------------------References---------------------------------
[1] Sauer, U., Lasko, D. R., Fiaux, J., Hochuli, M., Glaser, R., Szyperski, T., Wüthrich, K., and Bailey, J. E. (1999) J. Bacteriol. 181, 6679-6688.
[2] Fischer, E., and Sauer, U. (2003) Eur. J. Biochem. 270, 880-891. http://doi.org/dg3q64
[3] Fischer, E., Zamboni, N., and Sauer, U. (2004) Anal. Biochem. 325, 308-316. http://doi.org/c6kx35

Data file
Downloadfluxes KIMODATAID116_v2.xlsx
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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 2018-08-22 22:25:31 UTC

Updated 2018-08-22 22:28:56 UTC

Version 2

Status (reviewed) 2018-08-22 22:25:40 UTC




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