General Information info
|Manuscript title||Generating short-term kinetic responses of primary metabolism of Penicillium chrysogenum through glucose perturbation in the bioscope mini reactor.|
|Authors||U. Nasution, W.M. Van Gulik, A. Proell, W.A. van Winden, J.J. Heijnen|
|Affiliations||Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, Netherlands|
|Keywords||glucose pulse, in vivo kinetics, metabolome, Penicillium chrysogenum|
|Full text article||Nasution_2006.pdf|
|Project name||not specified|
Experiment Description info
|Data type||time-series data of metabolites|
|Execution date||not specified|
Experimental Details info
|Dilution rate (h-1)||0.05|
|Working volume (L)||4.0|
|Biomass concentration (g/L)||6.21 ± 0.16|
16.5 g/L glucose·H2O, 1 g/L KH2PO4, 5 g/L (NH4)2SO4, 0.5 g/L MgSO4·7H2O, and 10 ml/L of trace element solution. The trace element solution contained 15 g/L Na2-EDTA·2H2O, 0.5 g/L CuSO4·5H2O, 2 g/L ZnSO4·7H2O, 2 g/L MnSO4·H2O, 4 g/L FeSO4·7H2O and 0.5 g/L CaCl2·2H2O. For penicillin-G production the side chain precursor phenylacetic acid (PAA) was added at a concentration of 0.94 g/L.
|General protocol information||
rapid sampling of broth for measurement of intracellular metabolites was carried out at 5, 10, 15, 20, 25, 35, 50, 70 and 90 s after injection of the glucose solution.
Quenching procedure: 60 w/w % analytical grade methanol (ARCOS, Geel, Belgium) and water purified in a milli-QUFplus system (Millipore, Bedford, MA), buffered with 10 mM HEPES (Merck, Darmstadt, Germany) and adjusted to pH 7.5 with 3M KOA (Baker, Deventer, The Netherlands).
Extraction technique: hot ethanol
Sample analyzing method: HPLC-UV/RI, LC-ESI-MS
|Methods description - Notes||
Chemostat pulse experiments - for the pulse response experiment carried out in the chemostat 16 ml of a 125 g/L glucose solution was injected into the reactor within 1 s. This increased the residual glucose concentration to approximately 0.5 g/L. Subsequently, rapid sampling of broth for measurement of intracellular metabolites was carried out at 5, 10, 15, 20, 25, 35, 50, 70 and 90 s after injection of the glucose solution. Rapid sampling and quenching was carried out as described previously . Sampling times were chosen similar to the sampling times applied for the BioScope experiments. Rapid sampling for determination of extracelluar metabolite concentrations was carried out according to Mashego et al. .
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