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General Information info

Manuscript title Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis.
PubMed ID 19917605
Journal The Journal of Biological Chemistry
Year 2009
Authors Roelco J. Kleijn, Joerg M. Buescher, Ludovic Le Chat, Matthieu Jules, Stephane Aymerich and Uwe Sauer
Affiliations Institute of Molecular System Biology, ETH Zurich, CH-8093 Zurich, Switzerland
Keywords Metabolism, Metabolism/Gluconeogenesis, Metabolism/Intermediary, Metabolism/Regulation, Metabolism/Tricarboxylic Acid Cycle, Methods/Mass Spectrometry, flux analysis, metabolomics
Full text article Downloadarticle Kleijn_2010.pdf
Project name not specified

Experiment Description info

Organism Bacillus subtilis
Strain Wild-type BSB168 trp+
Data type metabolites at steady-state
Data units mM
Execution date not specified

Experimental Details info

Temperature (0C) 37
pH not specified
Carbon source glucose, glucose + malate, malate
Culture mode batch
Process condition aerobic
Dilution rate (h-1) -
Working volume (L) 0.03
Biomass concentration (g/L) 0.5
Medium composition

M9 minimal medium (per liter): 8.5 g of Na2HPO4.2H20, 3 g of KH2PO4, 1 g of NH4Cl, 0.5 g of NaCl. The following components were sterilized separately and then added (per liter of final medium): 1 ml of 0.1 m CaCl2.2H2O, 1 ml of 1 m MgSO4.7H2O, 1 ml of 50 mm FeCl3.6H2O, and 10 ml of trace salt solution. The trace salts solution contained (per liter): 170 mg of ZnCl2, 100 mg of MnCl2.4H2O, 60.0 mg of CoCl2.6H2O, 60.0 mg of Na2MoO4.2H2O, and 43.0 mg CuCl2.2H2O. When preparing the medium, the base salts were added first followed by CalCl2, MgSO4, FeCl3, and finally the trace elements.

General protocol information Sampling method: Liquid culture was grown to OD650 of ~0.1, at which time it was transferred to filter culture as follows: for each filter culture, 5 mL of liquid culture was passed through an 82mm diameter round nylon filter and placed cell-side up onto a agarose plate

Quenching procedure: rapid centrifugation method where 1 ml of culture broth was transferred into a 1.5-ml tube and centrifuged for 15 s at 14,000×g in a tabletop centrifuge. The supernatant was decanted, and the pellet was frozen in liquid nitrogen.

Extraction technique: hot ethanol

Sample analyzing method: LC-MS, GC-TOF

Methods description - Notes

GC-TOF Workflow: Dried aliquots were derivatized with 15 μl of either TMS-reagent (N-methyl-N-(trimethylsilyl)trifluoroacetamide, Fluka, Buchs, Switzerland) or TBDMS reagent (N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide, Fluka), and aliquots of 5 μl were injected into a 30-m GC column (HP-5-MS, 30 m × 0.25 mm × 0.25 μm, Agilent) using a CIS4 injector (Gerstel, Mülheim an der Ruhr, Germany) and solvent venting. Derivatized metabolites were detected on a Pegasus 3D TOF mass spectrometer (Leco), with an acquisition rate of 40 Hz. Peak detection and assignment were performed with ChromaTOF software (Version 2.32, Leco). Liquid Chromatography-MS/MS Workflow: Dry metabolite extracts were resuspended in 100 μl of water, 8 μl of which were injected on a Agilent 1100 series HPLC stack with a Synergi Hydro RP 2.1 × 150 × 4 column (Phenomenex, Aschaffenburg, Germany). Metabolites were detected using a 4000 QTRAP mass spectrometer (AB/MDS Sciex, Concord, Canada) operated in tandem MS mode with unit mass resolution. Analyst software (Version 1.4.2, AB/MDS Sciex) was used for both acquisition and integration. Ion spray voltage, auxiliary gas temperature, nebulizer gas, auxiliary gas, curtain gas, and collision gas were set to −4200 V, 650 °C, and 65, 40, 10, 4 (arbitrary units), respectively.
---------References---------
[1] Ewald J. C., Heux S., Zamboni N. (2009) Anal. Chem. 81, 3623–3629. http://doi.org/chgtcq [2] Büscher J. M., Czernik D., Ewald J. C., Sauer U., Zamboni N. (2009) Anal. Chem. 81, 2135–2143. http://doi.org/cjqg95

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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 2018-07-09 16:17:27 UTC

Updated 2018-07-11 22:28:32 UTC

Version 2

Status (reviewed) 2018-07-09 16:17:31 UTC




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