Summary of Study ST002381

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001531. The data can be accessed directly via it's Project DOI: 10.21228/M8ST4T This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002381
Study TitleRuegeria pomeroyi transporter mutant substrate drawdown
Study SummaryThe goal of this project was to identify bacterial transporters responsible for uptake of environmentally relevant marine metabolites. We used the model marine heterotrophic bacterium Ruegeria pomeroyi DSS-3, for which an arrayed library of single gene knockout mutants has been generated by selecting isolated from a barcoded transposon mutant library (BasSeq). Knockout mutants of putative transporters were grown on minimal medium with a single substrate as sole carbon source. Mutant defect was assessed by comparing the substrate drawdown of isolated mutants to drawdown by a pooled mutant library (BarSeq), a proxy for wildtype fitness.
Institute
University of Georgia
LaboratoryMoran Lab, Edison Lab
Last NameUchimiya
First NameMario
Address315 Riverbend Rd, Athens, GA, 30602, USA
Emailmario.uchimiya@uga.edu
Phone‭(706) 542-8387‬
Submit Date2022-11-16
Raw Data AvailableYes
Raw Data File Type(s)fid
Analysis Type DetailNMR
Release Date2022-12-27
Release Version1
Mario Uchimiya Mario Uchimiya
https://dx.doi.org/10.21228/M8ST4T
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001531
Project DOI:doi: 10.21228/M8ST4T
Project Title:Ruegeria pomeroyi transporter mutant substrate drawdown
Project Summary:The goal of this project was to identify bacterial transporters responsible for uptake of environmentally relevant marine metabolites. We used the model marine heterotrophic bacterium Ruegeria pomeroyi DSS-3, for which an arrayed library of single gene knockout mutants has been generated by selecting isolated from a barcoded transposon mutant library (BasSeq). Knockout mutants of putative transporters were grown on minimal medium with a single substrate as sole carbon source. Mutant defect was assessed by comparing the substrate drawdown of isolated mutants to drawdown by a pooled mutant library (BarSeq), a proxy for wildtype fitness.
Institute:University of Georgia
Laboratory:Moran Lab, Edison Lab
Last Name:Uchimiya
First Name:Mario
Address:315 Riverbend Rd, Athens, GA, 30602, USA
Email:mario.uchimiya@uga.edu
Phone:‭(706) 542-8387‬
Funding Source:Simons Foundation (542391), NSF (OCE-2019589)
Contributors:William F. Schroer

Subject:

Subject ID:SU002470
Subject Type:Bacteria
Subject Species:Ruegeria pomeroyi

Factors:

Subject type: Bacteria; Subject species: Ruegeria pomeroyi (Factor headings shown in green)

mb_sample_id local_sample_id Factor_1 (substrate) Factor_2 (bacteria) Factor_2 (time)
SA2374672533-OH butyrate none T0
SA2374682543-OH butyrate none T0
SA2374692593-OH butyrate none Tfinal
SA2374702603-OH butyrate none Tfinal
SA2374713263-OH butyrate pooled-BarSeq Tfinal
SA2374722633-OH butyrate pooled-BarSeq Tfinal
SA2374732753-OH butyrate pooled-BarSeq Tfinal
SA2374742673-OH butyrate ΔSPO2573 Tfinal
SA2374752793-OH butyrate ΔSPO2573 Tfinal
SA2374763743-OH butyrate ΔSPO2573 Tfinal
SA237547456azelaic acid none T0
SA237548455azelaic acid none T0
SA237549477azelaic acid none Tfinal
SA237550476azelaic acid none Tfinal
SA237551544azelaic acid pooled-BarSeq Tfinal
SA237552543azelaic acid pooled-BarSeq Tfinal
SA237553545azelaic acid pooled-BarSeq Tfinal
SA237554589azelaic acid ΔSPO1514 Tfinal
SA237555588azelaic acid ΔSPO1514 Tfinal
SA237556590azelaic acid ΔSPO1514 Tfinal
SA237477106Azeliac none T0
SA237478105Azeliac none T0
SA237479169Azeliac none Tfinal
SA237480170Azeliac none Tfinal
SA237481120Azeliac pooled-BarSeq Tfinal
SA237482122Azeliac pooled-BarSeq Tfinal
SA237483121Azeliac pooled-BarSeq Tfinal
SA237484133Azeliac ΔSPO1514 Tfinal
SA237485132Azeliac ΔSPO1514 Tfinal
SA237486134Azeliac ΔSPO1514 Tfinal
SA237487302Blank none Tfinal
SA237488502Blank none Tfinal
SA237489261Blank none Tfinal
SA237490273Blank none Tfinal
SA237491501Blank none Tfinal
SA237557448cadaverine none T0
SA237558447cadaverine none T0
SA237559468cadaverine none Tfinal
SA237560469cadaverine none Tfinal
SA237561532cadaverine pooled-BarSeq Tfinal
SA237562531cadaverine pooled-BarSeq Tfinal
SA237563533cadaverine pooled-BarSeq Tfinal
SA237564576cadaverine ΔSPO3469 Tfinal
SA237565577cadaverine ΔSPO3469 Tfinal
SA237566578cadaverine ΔSPO3469 Tfinal
SA237567406carnitine none T0
SA237568405carnitine none T0
SA237569419carnitine none Tfinal
SA237570420carnitine none Tfinal
SA237571312carnitine pooled-BarSeq Tfinal
SA237572311carnitine pooled-BarSeq Tfinal
SA237573310carnitine pooled-BarSeq Tfinal
SA237574380carnitine ΔSPO2995 Tfinal
SA237575381carnitine ΔSPO2995 Tfinal
SA237576379carnitine ΔSPO2995 Tfinal
SA237577382carnitine ΔSPO2996 Tfinal
SA237578383carnitine ΔSPO2996 Tfinal
SA237579384carnitine ΔSPO2996 Tfinal
SA237580453choline none T0
SA237581454choline none T0
SA237492104Choline none T0
SA237493103Choline none T0
SA237582475choline none Tfinal
SA237583474choline none Tfinal
SA237494167Choline none Tfinal
SA237495166Choline none Tfinal
SA237584540choline pooled-BarSeq Tfinal
SA237585541choline pooled-BarSeq Tfinal
SA237586542choline pooled-BarSeq Tfinal
SA237496119Choline pooled-BarSeq Tfinal
SA237497117Choline pooled-BarSeq Tfinal
SA237498118Choline pooled-BarSeq Tfinal
SA237587585choline ΔSPO1087 Tfinal
SA237588586choline ΔSPO1087 Tfinal
SA237589587choline ΔSPO1087 Tfinal
SA237499131Choline ΔSPO1087 Tfinal
SA237500129Choline ΔSPO1087 Tfinal
SA237501130Choline ΔSPO1087 Tfinal
SA237590250citrate none T0
SA237591249citrate none T0
SA237592346citrate none Tfinal
SA237593347citrate none Tfinal
SA237594333citrate none Tfinal
SA237595332citrate none Tfinal
SA237596370citrate none Tfinal
SA237597331citrate none Tfinal
SA237598255citrate none Tfinal
SA237599348citrate none Tfinal
SA237600371citrate none Tfinal
SA237601372citrate none Tfinal
SA237602323citrate pooled-BarSeq Tfinal
SA237603324citrate pooled-BarSeq Tfinal
SA237604322citrate pooled-BarSeq Tfinal
SA237605393citrate ΔSPO0184 Tfinal
SA237606392citrate ΔSPO0184 Tfinal
SA237607391citrate ΔSPO0184 Tfinal
SA237608439cysteate none T0
SA237609440cysteate none T0
SA237610462cysteate none Tfinal
SA237611461cysteate none Tfinal
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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:

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
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