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Manuscript title Modeling and simulation of the main metabolism in Escherichia coli and its several single-gene knockout mutants with experimental verification.
PubMed ID 21092096
Journal Microbial Cell Factories
Year 2010
Authors Tuty AA Kadir, Ahmad A Mannan, Andrzej M Kierzek, Johnjoe McFadden and Kazuyuki Shimizu
Affiliations Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502, Japan
Keywords Specific Growth Rate, Flux Balance Analysis, Oxidative Pentose Phosphate Pathway, Main Metabolic Pathway, Isotopomer Distribution
Full text article Downloadarticle Kadir_2010.pdf
Project name not specified

Experiment Description info

Organism Escherichia coli
Strain K-12 BW25113 and ppc, pck, pyk mutants
Data type flux measurements
Data units (mmol/g-dry cell weight/h)
Execution date not specified

Experimental Details info

Temperature (0C) 37
pH 7.0
Carbon source glucose,
Culture mode chemostat
Process condition aerobic
Dilution rate (h-1) 0.2
Working volume (L) 1.0
Biomass concentration (g/L) not specified
Medium composition

M9 sythetic medium: 48 mM Na2HPO4, 22 mM KH2PO4, 10 mM NaCl, 40 mM (NH4)zSO4 and 4 g/l of glucose. Components filter-sterilized separatory and then added (per liter of final volume): 1 ml 1 M MgSO4, 1 ml vitamin B1 (1 mgl stock), 1 ml 0.1 mM CaCl2, and 10 ml trace element solution containing (per liter): 0.55g CaCl2 1g FeCl3, 0.1 mg/l MnCl2.4H2O, 0.17 g ZnCl2, 0.043 g CuCl2.2H2O, 0.06 g CoCl2.2H2O, and 0.06 g Na2MoO4.2H2O).

General protocol information Flux analysis method: 13C constrained MFA

Platform: GC-MS

Methods description - Notes

13C-labeling experiments and sample preparation: The biomass sample was kept on ice for 2-3 minutes, and the sample was centrifuged at 6,000 rpm at 2°C for 15 minutes [1]. The cell pellets were washed three times with 20 mM Tris-HCl at pH 7.6 and suspended in 10 ml of 6 M HCl. The mixture was then hydrolyzed at 105°C for 15 hours in a sealed glass tube. During acid hydrolysis, tryptophan and cysteine were oxidized, and glutamine and asparagine were deaminated. The hydrolysate was evaporated to dryness. The dried material was dissolved in milli-Q water and filtered through a 0.22μm pore-size filter and evaporated to dryness. About 1.5 ml acetonitrile was added in the dried hydrolyte and incubated at room temperature overnight. After the color of liquid turned a color of pale yellow, it was filtrated through 0.22μm pore-size filter. The filtrate was then derivatized by N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBST-FA) (Aldrich, USA) at 110C for 30 minutes and was transferred to GC-MS sample tube for analysis [1]. 13C-labeling experiments were initiated after the culture reached steady state, which was inferred from the stable oxygen and carbon dioxide concentrations in the off-gas and stable OD in the effluent medium for at least twice as long as the residence time. The feed medium with 4 g/l of unlabeled glucose was then replaced by an identical medium containing 0.4 g of [U-13C] glucose, 0.4 g of [1-13 C] glucose, and 3.2 g of naturally labeled glucose per liter. Biomass samples for GC-MS analysis were taken after one residence time. Sample preparation, analytical procedures for GC-MS analysis, and flux computation are given elsewhere [2-4, 1]. Preparation of biomass hydrolysates and recording of the GC-MS spectra (PerklinElmer, Germany) were made as described previously [1,5,6].
--------------------------------------------References--------------------------------------
[1] Zhao J, Shimizu K. J Biotechnol. 2003, 101: 101-117. 17-23. http://doi.org/cdjhrn
[2] Peng L, Arauzo-Bravo MJ, Shimizu K. FEM Microbiology Letters. 2004, 235. http://doi.org/bn5mg6
[3] Siddiquee KAZ, Arauzo-Bravo MJ, Shimizu K. Appl Microbiol Biotechnol. 2004, 63: 407-417. http://doi.org/b9cm56 [4] Siddique KAZ, Arauzo-Bravo MJ, Shimizu K. FEMS Microbiology Letters. 2004, 235: 25-33. http://doi.org/dbcmv7 [5] Zhao J, Baba T, Mori H, Shimizu K. Metabolic Eng. 2004, 6: 164-174. http://doi.org/fxbfxd [6] Zhao J, Baba T, Mori H, Shimizu K. FEMS Microb Lett. 2003, 220: 295-301. http://doi.org/b3bb42

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Submission and curation info

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 2018-07-21 11:27:22 UTC

Updated 2018-07-21 11:37:43 UTC

Version 1

Status (reviewed) 2018-07-21 11:38:32 UTC




Associated Models


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EntryID
Model name Category Model Type Data used for Access Json
41
Authors: Hiroyuki Kurata and Yurie Sugimoto

Original paper: Improved kinetic model of Escherichia coli central carbon metabolism in batch and continuous cultures

Continuous kinetic model of Escherichia coli Metabolism ordinary differential equations Model building and Model validation Visto4 {"affiliation":"Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680 -4 Kawazu, Iizuka, Fukuoka 820 -8502, Japan","article_file_name":null,"authors":"Hiroyuki Kurata and Yurie Sugimoto","biomodels_id":"","category":"Metabolism","combine_archive_content_type":null,"combine_archive_file_name":null,"combine_archive_file_size":null,"combine_archive_updated_at":null,"comments":"Kinetic model source: http://www.cadlive.jp/cadlive_main/Softwares/KineticModel/Ecolimetabolism.html.","control":"2018-07-24T15:46:04Z","dilution_rate":"0.2, 0.4, 0.5 and 0.7","id":41,"journal":"Journal of Bioscience and Bioengineering","keywords":"Kinetic model, Synthetic biology, Central carbon metabolism, Dynamic simulation, Escherichia coli","main_organism":"Escherichia coli","manuscript_title":"Improved kinetic model of Escherichia coli central carbon metabolism in batch and continuous cultures","model_name":"Continuous kinetic model of Escherichia coli","model_type":"ordinary differential equations","organism_id":null,"project_name":"","pubmed_id":"29054464","review_journal_id":null,"sbml_file_name":"Kurata_2018.pdf","software":"MATLAB (MathWorks)","used_for":"---\n- Model building\n- Model validation\n","year":2018} Administrator KiMoSys



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