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MB Sample ID: SA237588
| Local Sample ID: | 586 |
| Subject ID: | SU002470 |
| Subject Type: | Bacteria |
| Subject Species: | Ruegeria pomeroyi |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002470 |
| Subject Type: | Bacteria |
| Subject Species: | Ruegeria pomeroyi |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 586 | SA237588 | FL029479 | choline | Factor_1 (substrate) |
| 586 | SA237588 | FL029479 | ΔSPO1087 | Factor_2 (bacteria) |
| 586 | SA237588 | FL029479 | Tfinal | Factor_2 (time) |
Collection:
| Collection ID: | CO002463 |
| Collection Summary: | Cultures, 220 ul in 96 well plate (Fisher), were centrifuged at 2250 xg for 10 minutes to pellet cells. Supernatant, 200 ul, was collected and transferred to new 96 well plate. Samples were stored at -80 oC until further processing. |
| Collection Protocol Filename: | 2_Collection protocol_UGA_mutant_Nov2022.docx |
| Sample Type: | Artificial sea water |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002482 |
| Treatment Summary: | Mutants of Ruegeria pomeroyi DSS-3 were grown on minimal medium containing a single substrate as sole carbon source, they were screened for their ability to drawdown this target substrate. Screens were performed in L1 minimal medium modified to a salinity of 20 and amended with ammonium (3 mM) and kanamycin (50 ug ml-1). For each mutant-substrate pair, 3 replicate 220 µl cultures were prepared in 96 well plates by inoculating 3 ul of washed (3x) overnight mutant cultures into minimal medium containing the candidate substrate at 8 mM carbon concentration. Cultures were grown shaking at 25oC for 24 h or 36 h, depending on the growth rate supported by the carbon source. As a positive control, four wells with the same medium were inoculated with washed overnight cultures of the pooled-BarSeq library, used as a proxy for wild-type R. pomeroyi fitness but harboring a transposon/kanamycin resistance gene insertion. |
| Treatment Protocol Filename: | 3_Treatment protocol_UGA_mutant_Nov2022.docx |
Sample Preparation:
| Sampleprep ID: | SP002476 |
| Sampleprep Summary: | Samples were thawed on ice. Each sample, 180 ul, was transferred to a 1.5 ml microcentrifuge tube and mixed with 20 µL of a deuterated phosphate buffer (30 mmol L-1, pH 7.4) and an internal standard 2,2-dimethyl-2-silapentane-5-sulfonate-d6 (DSS, 1 mmol L-1) and transferred to a 3-mm NMR tube (Bruker). |
| Sampleprep Protocol Filename: | 4_Sample preparation protocol_UGA_mutant_Nov2022.docx |
Analysis:
| MB Sample ID: | SA237588 |
| Analysis ID: | AN003880 |
| Analysis Type: | NMR |
| Analysis Protocol File: | 5_Analysis protocol_UGA_mutant_Nov2022.docx |
| Num Factors: | 118 |
| Num Metabolites: | 21 |
| Units: | intensity_(peak_area) |
NMR:
| NMR ID: | NM000256 |
| Analysis ID: | AN003880 |
| Instrument Name: | Bruker |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | 1D-1H |
| NMR Comments: | Analysis protocol: Instrument – Metabolites were analyzed by nuclear magnetic resonance (NMR) spectroscopy using a Bruker Avance lll 600 MHz spectrometer equipped with a 5-mm TCI cryoprobe. Data acquisition– Data were acquired by a one dimensional 1H experiment with water suppression (noesypr1d, Bruker) at 298K using TopSpin 3.6.4 (Bruker). For only glycerol, 1H J-resolved experiment (jresgpprqf) was used to avoid overlapping background peaks. Acquisition parameters are in ‘6_Acquisition and processing parameters_UGA_mutant_Nov2022.xlsx. Specific pulse programs used for individual samples are in ‘1_Study design_UGA_mutant_Nov2022.xlsx. Data processing – The raw Bruker spectra were processed using NMRPipe on NMRbox. For Jres, spectra were further symmetrized and tilted. Detailed spectrum processing parameters for individual NMR experiments are in ‘6_Acquisition and processing parameters_UGA_mutant_Nov2022.xlsx. NMRPipe scripts are available in folder ‘Data_analysis’. Downstream data analysis: Downstream analysis was conducted using Metabolomics Toolbox (https://github.com/artedison/Edison_Lab_Shared_Metabolomics_UGA) and MATLAB R2022a (MathWorks). All the input files, processing steps and scripts, and the output files are available in folder ‘Data_analysis’. |
| Spectrometer Frequency: | 600 MHz |
