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Manuscript title Catching prompt metabolite dynamics in Escherichia coli with the BioScope at oxygen rich conditions.
PubMed ID 20447466
Journal Metabolic Engineering
Year 2010
Authors Marjan De Mey, Hilal Taymaz-Nikerel, Gino Baart, Hendrik Waegeman, Jo Maertens, Joseph J. Heijnen, Walter M. Van Gulik
Affiliations Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium and Department of Biotechnology, Delft University of Technology, Kluyver Center for Genomics of Industrial Fermentation, 2628 BC Delft, The Netherlands
Keywords Escherichia coli, glucose pulse, BioScope, chemostat
Full text article Downloadarticle DeMey_2010.pdf
Project name not specified

Experiment Description info

Organism Escherichia coli
Strain K-12 MG1655
Data type metabolites at steady-state
Data units (µmol/gDW)
Execution date not specified

Experimental Details info

Temperature (0C) 37.0
pH 5.0
Carbon source glucose,
Culture mode chemostat
Process condition aerobic
Dilution rate (h-1) 0.1
Working volume (L) 4.0
Biomass concentration (g/L) 8.4 (Experiment 1) and 8.02 (Experiment 2)
Medium composition

The composition of the low Cl- minimal medium was, per liter: 1.25g(NH4)2SO4, 1.15g KH2PO4, 0.5g MgSO4.7H2O, 0.5g NaCl, 30g glucose.1H2O, 0.001 gthiamine–HCl, 2ml of trace elements solution and 0.2 ml silicone-based antifoaming agent (BDH,Poole,UK). The composition of the trace elements solution was described in Verduyn et al. [1].

[1] Verduyn, C., Postma, E., Scheffers, W.A., van Dijken, J.P., 1992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8 (7), 501–517

General protocol information Sampling method: 1ml of broth rapidly withdrawn from the bioreactor.

Quenching procedure: tubes containing 5ml of 60% aqueous methanol, pre-cooled at -40 ºC and immediately mixed after sampling by vortexing.

Extraction technique: boiling ethanol

Sample analyzing method: LC-ESI-MS

Methods description - Notes

Metabolite extraction procedure - metabolites were extracted in 75 % boiling ethanol (3 min, 90 ºC) as described in Taymaz-Nikerel et al. [1]. Before extraction, 100 µl of 100% U–13C-labeled cell extract was added as internal standard.
Measurement of intracellular metabolite concentrations - metabolites of the glycolysis, TCA cycle and PPP were quantified with Isotope Dilution Mass Spectrometry (IDMS) as described by van Dam et al. [2] and Wu et al. [3]. The concentrations of the nucleotides were also analyzed with IDMS. Further details of the applied LC–ESI–MS/MS procedure have been described elsewhere [4].

[1] Taymaz-Nikerel, H., deMey,M., Ras,C., tenPierick,A., Seifar,R.M., vanDam,J.C., Heijnen, J.J., vanGulik,W.M.,2009. Development and application of a differential method for reliable metabolome analysisin Escherichia coli. Analytical Biochemistry 386 (1), 9–19.
[2] van Dam,J.C.,Eman,M.R.,Frank,J.,Lange,H.C.,vanDedem,G.W.K., Heijnen,S.J., 2002. Analysis of glycolytic intermediates in Saccharomyces cerevisiae using anion exchange chromatography and electrospray ionization with tandem mass spectrometric detection. Analytica Chimica Acta 460 (2), 209–218.
[3] Wu, L.,Mashego,M.R.,vanDam,J.C.,Proell,A.M.,Vinke,J.L.,Ras,C., van Winden, W.A., vanGulik,W.M.,Heijnen,J.J.,2005.Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13C-labeled cell extracts as internal standards. Analytical Biochemistry 336, 164–171.
[4] Seifar, R.M.,Ras,C., vanDam,J.C.,van Gulik,W.M., Heijnen,J.J., van Winden,W.A., 2009. Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry. Analytical Biochemistry 388 (2), 213–219.

Data file
Downloadmetabolites KIMODATAID74_v0.xlsx
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Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Interests: mathematical modeling, accessible data, use of data

Created 2013-06-25 14:31:12 UTC

Updated 2014-06-13 17:21:23 UTC

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

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