General Information info
Experiment Description info
|Strain||M145 (W.T.) and phoP mutant (INB201)|
|Data type||time-series data of metabolites|
|Execution date||not specified|
Experimental Details info
|Dilution rate (h-1)||—|
|Working volume (L)||1.8|
|Biomass concentration (g/L)||see worksheets|
(SSBM-P) medium for studying the effect of phosphate depletion: Na-glutamate, 55.2 g/L; D-glucose, 40 g/L; MgSO4, 2.0 mM; phosphate, 4.6 mM; supplemented minimal medium trace element solution SMM-TE , 8 mL/L and TMS1, 5.6 mL/L. TMS1 consisted of FeSO4 × 7 H2O, 5 g/L; CuSO4 × 5 H2O, 390 mg/L; ZnSO4 × 7 H2O, 440 mg/L; MnSO4 × H2O, 150 mg/L; Na2MoO4 × 2 H2O, 10 mg/L; CoCl2 × 6 H2O, 20 mg/L, and HCl, 50 ml/L. (SSBM-E) medium for studying the effect of L-glutamate depletion: identical to SSBM-P except for the concentrations of Na-glutamate and phosphate adjusted to be 15 g/L and 9.2 mM, respectively.
|General protocol information||
Samples for metabolite profiling were withdrawn from the cultivations in 1–2 h time intervals.
Quenching procedure: Quenching procedure: 5 mL culture sample was withdrawn from the fermentation vessel and immediately applied to a 0.8 μm Supor 800 filter (Pall). On the filter disc, the cells were subsequently washed twice with one volume 2.63% (w/v) NaCl solution each.
Extraction technique: methanol
Sample analyzing method: GC-MS, LC-MS
|Methods description - Notes||
Metabolite Extraction - samples stored at −80 °C were completely thawed on an ethanol bath at −23 °C. An internal standard mix was added to each 25 mL sample (biomass from 5 mL sample on filter in 25 mL 60% methanol solution) yielding final concentrations of 3.34 mM D3-alanine, 312.5 μM D4-succinate, 1.67 μM D8-valine, 62.5 μM 13C6-glucose, 0.416 μM 13C10, 15N5-adenosine monophosphate and 1.04 μM 13C1-α-ketoisocaproic acid). This standard mix included compounds to be used as internal standards in different analytical methods for metabolites with different chemical properties (organic acids, phosphometabolites, sugars). Samples were then subjected to three cycles of freezing on liquid nitrogen and thawing at −23 °C on the ethanol bath, found to be sufficient for reaching a maximum of compound extraction into the 60% methanol, and thereafter centrifuged for 5 min at −9 °C and 6000 × g. Supernatants were transferred to new tubes pre-chilled at −23 °C and then divided into aliquots á 6 mL in 15 mL screw cap tubes for analysis using different metabolite profiling methods. Samples were frozen at −80 °C and subsequently subjected to solvent evaporation on a freeze-dryer for 24 h. The freeze-dried samples were stored at −80 °C until MS analysis.
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