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MB Sample ID: SA094558
| Local Sample ID: | 9404 +Gly 3 |
| Subject ID: | SU001382 |
| Subject Type: | Bacteria |
| Subject Species: | Salmonella enterica serovar Typhimurium |
| Taxonomy ID: | 90371 |
| Genotype Strain: | LT2 |
| Gender: | Not applicable |
| Subject Comments: | The wild type as describe, also used a ridA null mutant (DM3480, ridA3::MudJ1734), DM9404 |
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Subject:
| Subject ID: | SU001382 |
| Subject Type: | Bacteria |
| Subject Species: | Salmonella enterica serovar Typhimurium |
| Taxonomy ID: | 90371 |
| Genotype Strain: | LT2 |
| Gender: | Not applicable |
| Subject Comments: | The wild type as describe, also used a ridA null mutant (DM3480, ridA3::MudJ1734), DM9404 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 9404 +Gly 3 | SA094558 | FL013495 | wt_gly | Sample_group |
| 9404 +Gly 3 | SA094558 | FL013495 | wild-type | Genotype |
| 9404 +Gly 3 | SA094558 | FL013495 | glycine | Suplement |
Collection:
| Collection ID: | CO001377 |
| Collection Summary: | Bacterial cells were grown in media. Each sample was centrifuged and the spent media was collected as well as the pelleted bacterial cells, both flash frozen in liquid nitrogen. |
| Collection Protocol Filename: | Cells_NMR_AJB_12_16_2018 |
| Sample Type: | Bacterial cells |
| Collection Method: | Centrifuge and freeze |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001397 |
| Treatment Summary: | Ten biologically independent wild-type and ridA mutant cultures were grown overnight in NB medium shaking at 37 °C and used to inoculate (1% inoculum) 250 mL non-baffled flasks holding 125 mL of medium. Each culture inoculated one each of the three media types (minimal medium, minimal medium with 1 mM L-isoleucine, and minimal medium with 1 mM glycine), for a total of 60 flasks. Flasks were randomly arranged in an Innova®44 incubator and cultures allowed to grow 16 h at 37 °C, shaking at 180 RPM. Cultures were chilled 5 min on ice and then harvested by centrifugation at 7,000 x G for 10 min at 4 °C. The supernatant was decanted, with 10 mL transferred to sterile 15 mL conical tubes and flash-frozen using liquid nitrogen for downstream analysis of external metabolites. Cell pellets were transferred to sterile 15 mL conical tubes after resuspension in 10 mL ddH2O, prior to a second pelleting at 7,000 x G for 10 min at 4 °C. The supernatant was decanted and pellets were flash-frozen using liquid nitrogen and stored at -80 °C. |
Sample Preparation:
| Sampleprep ID: | SP001390 |
| Sampleprep Summary: | Preparation of growth medium samples. Spent media from each bacterial culture was lyophilized (VirTis Benchtop K) for 48 h. Once dry, each lyophilized sample was reconstituted in 150 L of 100 mM sodium phosphate buffer (Cambridge Isotope Laboratories), pH 7.0, containing 1/3 mM DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid, Cambridge Isotope Laboratories) as an internal standard. Each sample was centrifuged at 20,000 X G for 30 min and 50 L of supernatant was transferred by a Bruker SamplePro liquid handler into 1.7 mm SampleJet NMR tubes (Bruker Biospin). Metabolite extraction from bacterial pellets. Each frozen bacterial pellet was thawed on ice and 1 mL of ice cold 80/20 methanol/water together with approximately 200 mL of 0.7mm silica beads (BioSpec products). Homogenization was carried out using a FastPrep 96 (MPBIO). The samples and extraction blanks went through three cycles of homogenization at 1800 rpm for 300 s each. At the end of each cycle samples and controls were centrifuged at 20000 x G for 30 min. Each supernatant was transferred to a new tube and 1 mL of ice-cold methanol/water added to the original tubes before each new cycle. The combined supernatants from each cycle were pooled and concentrated overnight using a CentriVap Benchtop Vacuum Concentrator (Labconco) down to 0.1 mL. The samples were then diluted with 0.5 mL of methanol/water and transferred into 0.6 mL centrifuge tube and concentrated to dryness. The extracts were reconstituted in 150 uL of deuterated 100 mM sodium phosphate buffer containing 1/3 mM of the internal standard DSS (d6 4,4-dimethyl-4-silapentane-1-sulfonic acid) at pH 7.0 and vortex mixed for 5 min. Each sample was centrifuged at 20000 x G for 30 min and transferred by a Bruker SamplePro liquid handler into 1.7 mm SampleJet NMR tubes. Extraction blanks were prepared following the same procedure except the biological material was replaced with an equal volume of water. Solvent blanks consisted of the reconstituting NMR buffer (deuterated sodium phosphate buffer with DSS). |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | -80℃ |
Analysis:
| MB Sample ID: | SA094558 |
| Analysis ID: | AN002177 |
| Laboratory Name: | Edison Lab - Complex Carbohydrate Research Center |
| Analysis Type: | NMR |
| Acquisition Date: | jan-4-2019 |
| Operator Name: | Goncalo Gouveia |
| Detector Type: | FT-NMR |
| Num Factors: | 6 |
| Num Metabolites: | 21 |
| Units: | Area under the curve |
NMR:
| NMR ID: | NM000160 |
| Analysis ID: | AN002177 |
| Instrument Name: | 600Mhz Avance III HD |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | 1D-1H |
| Spectrometer Frequency: | 600 |
| NMR Probe: | TCI - cryo-probe |
| NMR Solvent: | D2O |
| NMR Tube Size: | 1.7 mm |
| Shimming Method: | TopShim |
| Pulse Sequence: | PROJECT (periodic refocusing of J evolution by coherence transfer) |
| Water Suppression: | presat |
| Receiver Gain: | 203 |
| Number Of Scans: | 64 |
| Dummy Scans: | 16 |
| Acquisition Time: | 1.3107200 |
| Spectral Width: | 20.8 ppm |
| Num Data Points Acquired: | 32768 |
| Line Broadening: | 2 |
| Apodization: | Exponential |
| Baseline Correction Method: | Polynomial order 3 |
| Chemical Shift Ref Std: | 0.00 ppm |
