DataEntryID 88 General information Manuscript title: Transcriptional regulation is insufficient to explain substrate-induced flux changes in Bacillus subtilis PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/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: https://kimosys.org/rails/active_storage/blobs/eyJfcmFpbHMiOnsibWVzc2FnZSI6IkJBaHBBcWtFIiwiZXhwIjpudWxsLCJwdXIiOiJibG9iX2lkIn19--fc428f45b69f29a5074609f32b4098de97c3f590/Chubukov_2013.pdf Project name: not specified Experiment description Organism: Bacillus subtilis Strain: BSB168 Data type: metabolites at steady-state Data units: mMol/L cell volume Execution date: not specified Experimental details Temperature (°C): 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: 0.03 L 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: —; Extraction list: hot ethanol; Analysis list: HPLC-MS; Methods description: 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 Data file: http://kimosys.org/repository/88/download?parameter=1192; Alternative formats: no files uploaded Submission and curation Entered by: Administrator KiMoSys Created: 2015-05-05 16:47:23 UTC Updated: 2020-04-24 16:10:35 UTC Version: 3 Status: (reviewed) 2015-05-05 16:47:54 UTC Views: 183 Downloads: 51