General Information info
|Manuscript title||13C‐metabolic flux analysis for batch culture of Escherichia coli and its pyk and pgi gene knockout mutants based on mass isotopomer distribution of intracellular metabolites.|
|Authors||Yoshihiro Toya, Nobuyoshi Ishii, Kenji Nakahigashi, Takashi Hirasawa, Tomoyoshi Soga, Masaru Tomita, Kazuyuki Shimizu|
|Affiliations||Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan|
|Keywords||13C-metabolic flux analysis, batch culture, Escherichia coli, Pyk mutant, Pgi|
|Full text article||Toya_2010.pdf|
|Project name||not specified|
Experiment Description info
|Strain||Wild‐type E. coli BW25113, Pyk and Pgi mutants|
|Data type||time-series data of metabolites|
|Data units||g/L and mM|
|Execution date||not specified|
Experimental Details info
|Dilution rate (h-1)||—|
|Working volume (L)||1.2|
|Biomass concentration (g/L)||1 OD600 = 0.3 g/L|
Synthetic medium (48 mM Na2HPO4, 22 mM KH2PO4, 10 mM NaCl, 45 mM (NH4)2SO4, 4 g/L glucose) supplemented with 1 mM MgSO4, 1 mg/mL thiamine, 0.056 mg/L CaCl2, 0.08 mg/L FeCl3, 0.01 mg/L MnCl2·4H2O, 0.017 mg/L ZnCl2, 0.0043 mg/L CuCl2·2H2O, 0.006 mg/L CoCl2·2H2O, and 0.06 mg/L Na2MoO4·2H2O.
|General protocol information||
An aliquot of culture broth containing 0.015 g of cells was passed through a 0.45‐μm pore size filter.
Quenching procedure: cells washed with 20 mL of Milli‐Q water at 37°C and metabolism was stopped by submerging in 4 mL of methanol at 4°C containing 2 μM 2‐(N‐morpholino)ethanesulfonic acid, 2 μM trimesate, 2 μM methionine sulfone and 2 μM 3‐aminopyrrolidine as standard.
Extraction technique: chloroform-methanol
Sample analyzing method: CE-TOF-MS
|Methods description - Notes||
Extracellular metabolites: glucose and acetate, were measured by enzymatic assay kits (F‐kit, Roche Diagnostics, Germany). Intracellular metabolites: sample preparation was carried out using the modified method described by Ohashi et al.  An aliquot of culture broth containing 0.015 g of cells was passed through a 0.45‐μm pore size filter (Millipore). The cells on the filter were washed with 20 mL of Milli‐Q water at 37°C and metabolism was stopped by submerging in 4 mL of methanol at 4°C containing 2 μM 2‐(N‐morpholino)ethanesulfonic acid, 2 μM trimesate, 2 μM methionine sulfone and 2 μM 3‐aminopyrrolidine as internal standard. Quantities of 4 mL of chloroform and 1.6 mL of Milli‐Q water were added to the solution, which was then fully mixed. The solution was centrifuged at 2,300g for 5 min at 4°C, and the separated methanol layer was filtered by centrifugation through a Millipore 5‐kDa cutoff filter to remove high‐molecular‐weight compounds. The filtrate was lyophilized and then dissolved in 50 μL of Milli‐Q water before CE‐TOFMS analysis. CE‐TOFMS experiments were performed on an Agilent CE capillary electrophoresis system (Agilent Technologies, Germany) and an Agilent G3250AA LC/MSD TOF system (Agilent Technologies, Palo Alto, CA). The CE‐TOFMS conditions used for anionic metabolite analysis have been described elsewhere [2,3]. All measurements were performed three times.
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