Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA053777

Local Sample ID:	4A
Subject ID:	SU000952
Subject Type:	NMR based metabolomics of microbes
Subject Species:	Microbacterium sediminis
Taxonomy ID:	904291

Select appropriate tab below to view additional metadata details:

Sample Preparation:

Sampleprep ID:	SP000959
Sampleprep Summary:	All 100 mL of the fermentation broth in a flask was harvested and poured into a 250 mL centrifuge bottle and centrifuged at 6000g and 4 °C for 5 min. The supernatant was discarded and the cell pellets were quenched using 100 mL of a buffer composed of 3:2 methanol/water and 0.85% (wt./vol.) NaCl at -40 °C. The resuspended mixture was again centrifuged at 6000g and 4 °C for 5 min. The cell pellets were then washed using cold PBS for 3 times. The mixture was resuspended and transferred into a 5 mL Eppendorf tube during the 3rd wash and then centrifuged at 6000g and 4 °C for 5 min. The cell pellets were kept under -80 °C until use. Intracellular metabolites were extracted using a procedure adopted from Ye et al. (Ye et al. 2012). The frozen samples were homogenized in 600 μL of cold 1:1 acetonitrile/water buffer. To destroy the bacterial cells, the samples were further sonicated on wet ice for 180 cycles with each cycle consisting of 2 s pulses and 3 s stops. The supernatant was collected by centrifugation at 12000 g for 10 min at 4 °C. The remaining solid residues were further extracted using the same extract solution and intensively homogenized via vortexing. The second supernatant was collected after centrifugation and pooled with the first one. The combined supernatants from the two extractions were lyophilized, and stored at -80 °C. Immediately before 1H NMR measurements were taken, the extract powder was redisclosed in 550 µL phosphate buffer (50 mM K2HPO4/NaH2PO4, 10% D2O, 1mM 3-(Trimethylsilyl) propionate-2,2,3,3-d4 acid sodium salt (TSP), pH7.4). Subsequently, all the samples were vortexed and centrifuged at 12000 g for 15 min at 4 °C to remove any insoluble components. Finally, aliquots of the supernatant were transferred into 5 mm NMR tubes (Beckonert et al. 2007).

Sampleprep ID:

SP000959

Sampleprep Summary:

All 100 mL of the fermentation broth in a flask was harvested and poured into a 250 mL centrifuge bottle and centrifuged at 6000g and 4 °C for 5 min. The supernatant was discarded and the cell pellets were quenched using 100 mL of a buffer composed of 3:2 methanol/water and 0.85% (wt./vol.) NaCl at -40 °C. The resuspended mixture was again centrifuged at 6000g and 4 °C for 5 min. The cell pellets were then washed using cold PBS for 3 times. The mixture was resuspended and transferred into a 5 mL Eppendorf tube during the 3rd wash and then centrifuged at 6000g and 4 °C for 5 min. The cell pellets were kept under -80 °C until use. Intracellular metabolites were extracted using a procedure adopted from Ye et al. (Ye et al. 2012). The frozen samples were homogenized in 600 μL of cold 1:1 acetonitrile/water buffer. To destroy the bacterial cells, the samples were further sonicated on wet ice for 180 cycles with each cycle consisting of 2 s pulses and 3 s stops. The supernatant was collected by centrifugation at 12000 g for 10 min at 4 °C. The remaining solid residues were further extracted using the same extract solution and intensively homogenized via vortexing. The second supernatant was collected after centrifugation and pooled with the first one. The combined supernatants from the two extractions were lyophilized, and stored at -80 °C. Immediately before 1H NMR measurements were taken, the extract powder was redisclosed in 550 µL phosphate buffer (50 mM K2HPO4/NaH2PO4, 10% D2O, 1mM 3-(Trimethylsilyl) propionate-2,2,3,3-d4 acid sodium salt (TSP), pH7.4). Subsequently, all the samples were vortexed and centrifuged at 12000 g for 15 min at 4 °C to remove any insoluble components. Finally, aliquots of the supernatant were transferred into 5 mm NMR tubes (Beckonert et al. 2007).