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General Information info

Manuscript title Transcriptional regulation is insufficient to explain substrate-induced flux changes in Bacillus subtilis
PubMed ID 24281055
Journal Molecular Systems Biology
Year 2013
Authors Victor Chubukov, Markus Uhr, Ludovic Le Chat, Roelco J Kleijn, Matthieu Jules, Hannes Link, Stephane Aymerich, Joerg Stelling and Uwe Sauer
Affiliations Institute of Molecular System Biology, ETH Zurich, Zurich, Switzerland.
Keywords central carbon metabolism, metabolic flux, transcriptional regulation
Full text article Downloadarticle Chubukov_2013.pdf
Project name not specified

Experiment Description info

Organism Bacillus subtilis
Strain BSB168
Data type metabolites at steady-state
Data units mMol/L cell volume
Execution date not specified

Experimental Details info

Temperature (0C) 37.0
pH
Carbon source glucose, fructose, gluconate, succinate+glutamate, glycerol, malate, malate, malate+glucose, pyruvate
Culture mode batch
Process condition aerobic
Dilution rate (h-1)
Working volume (L) 0.03
Biomass concentration (g/L) 3.0 g cells/OD600
Medium composition

M9 minimal medium (per liter): 8.5 g Na2HPO4·2H2O, 3 g KH2PO4, 1 g NH4Cl, 0.5 g NaCl. The following components were sterilized separately and then added (per liter of final medium): 1 ml 0.1 M CaCl2, 1 ml 1 M MgSO4, 1 ml 50 mM FeCl3 and 10 ml trace salts solution. The trace salts solution contained (per liter): 170 mg ZnCl2, 100 mg MnCl2·4H2O, 60.0 mg CoCl2·6H2O, 60.0 mg Na2MoO4·2H2O and 43.0 mg CuCl2·2H2O. Filter‐sterilized carbon sources were added separately to the medium, pH neutralized with 4 M NaOH where necessary.

General protocol information Sampling method: Sampling method: Two samples for intracellular metabolite quantification were taken within 5 min of each other from the shake flask cultures during exponential growth at an OD600 between 0.8 and 1.2.

Quenching procedure:

Extraction technique: hot ethanol

Sample analyzing method: HPLC-MS

Methods description - Notes

In all, 2 ml of culture was vacuum filtered on a 0.45‐μm pore size nitrocellulose filter (Millipore) and immediately washed with two volumes of fresh M9 medium containing the respective carbon source and adjusted to the pH of the culture at the time of sampling. Sampling was performed in a room kept at 37°C. After washing, the filter was directly transferred for extraction into 4 ml of 60% (v/v) ethanol/water and kept at 78°C for 2 min. The metabolite extract was separated from cell debris and nitrocellulose by centrifugation at 14 000 g at 4°C for 10 min. The supernatants were dried at 0.12 mbar to complete dryness in a speed vac set‐up (Christ, Osterode am Harz, Germany). Dry metabolite extracts were stored at −80°C until analysis.
Metabolite concentrations were determined by using an ion‐pairing ultrahigh performance liquid chromatography‐tandem mass spectrometry method [1]. Dry metabolite extracts were resuspended in 100 μl, 10 μl of which was injected on a Waters Acquity UPLC with a Waters Acquity T3 end‐capped reverse phase column (150 × 2.1 mm × 1.8 μm; Waters Corporation, Milford, MA, USA). Metabolites were detected on a tandem mass spectrometer (Thermo TSQ Quantum Triple Quadropole with Electron‐Spray Ionization; Thermo Scientific, Waltham, MA, USA). --------------------------------------------References---------------------------------------
[1] Büscher JM, Moco S, Sauer U, Zamboni N (2010). Anal Chem 82: 4403–4412. http://doi.org/dsp5gb

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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 2015-05-05 16:47:23 UTC

Updated 2015-05-05 23:22:23 UTC

Version 3

Status (reviewed) 2015-05-05 16:47:54 UTC




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