Dataset: High Throughput Growth Screen Data

Name: High throughput growth screening of the marine bacterium Ruegeria pomeroyi DSS-3 knockout mutants on 70 environmentally relevant marine substrates done in 2021.
Published: 2023-07-17
Keywords: RB-TnSeq, gene fitness, transporters, metabolism, arrayed mutant library, genetic screen, oceans

Cite This Dataset

DOI:10.26008/1912/bco-dmo.904246.1

Temporal Extent: 2022-07-07 - 2022-09-29

Project:

Function and Importance of Marine Bacterial Transporters of Plankton Exometabolites (C-CoMP Marine Bacterial Transporters)

Program:

Center for Chemical Currencies of a Microbial Planet (C-CoMP)

Principal Investigator:

Mary Ann Moran (University of Georgia, UGA)

Co-Principal Investigator:

Christopher R. Reisch (University of Florida, UF)

Scientist:

Catalina Mejia (University of Florida, UF-SFRC)

Lidimarie Trujillo Rodriguez (University of Florida, UF)

Student:

William F. Schroer (University of Georgia, UGA)

Contact:

William F. Schroer (University of Georgia, UGA)

Data Manager:

Laura Gray (Woods Hole Oceanographic Institution, WHOI)

BCO-DMO Data Manager:

Karen Soenen (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2023-07-17

Restricted:

Validated:

Yes

Current State:

Final no updates expected

High throughput growth screening of the marine bacterium Ruegeria pomeroyi DSS-3 knockout mutants on 70 environmentally relevant marine substrates done in 2021.

Abstract:

An array of transporter knockout mutants of the marine bacterium Ruegeria pomeroyi DSS-3 mutants were screened for growth on 70 environmentally relevant marine substrates. The array contains 156 isolated knockout mutants of putative transporter genes as well as positive and negative growth controls and is distributed across two 96-well plates. Actively growing cultures were inoculated into freshly prepared 96 well plates containing minimal medium with a single substrates as sole carbon source. Plates were grown for 24 to 96 hours with growth monitored by optical density at 600 nm. Mutants that demonstrated growth defects on a given substrate, relative to typical growth on that substrate were selected for higher resolution follow up testing.

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Archival Copy

Version Date	Archive	Persistent Identifier (PID)	Date PID Issued
2023-07-17	Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)	10.26008/1912/bco-dmo.904246.1	2023-07-21

Methods & Sampling

The data were generated from laboratory experiments performed at the University of Florida, Gainesville, Florida.

Mutant cultures were pre-grown overnight in ½ YTSS medium with 50 mg ml-1 kanamycin. Screens were performed in L1 minimal medium (Guillard and Hargraves 1993) modified to a salinity of 20 ppt and amended with ammonium (3 mM), kanamycin (50 mg ml-1), and phosphorus as PO43- at 36 mM. Overnight cultures of individual mutants (2 ml) were inoculated into 198 ml of modified L1 with a single substrate as the sole carbon source at 8 mM carbon. Plates were incubated at 25oC with shaking, and optical density (OD600) was read at intervals of 6-24 h until cultures entered stationary phase at ~24-72 h (SpectraMax M3, Molecular Devices, San Jose, CA). Mutants exhibiting phenotypes in the initial screen were moved to the targeted screen.

Data Processing Description

Minimal data processing was performed. Data were imported into Excel merged and reformatted in R version 4.2.2.

A supplemental file is provided with alternate formatting, see supplemental file description for more information.

BCO-DMO Processing Notes:
* Added year to date column
* Converted date column to ISO format
* Removed row number column

Data Files

File
High Throughput Growth Screening filename: 904246_v1_hts.csv (Comma Separated Values (.csv), 8.45 MB) MD5:a9347feb56f410a34a8dba636a9d2889 Primary data file for dataset 904246, version 1.

Supplemental Files

File

High Throughput Growth Screening Data, alternat format
filename: HighThroughputScreenForBCODMO3_17.xlsx

(Microsoft Excel, 1.32 MB)
MD5:ceeae7fb88cc0aff9a73322dacaeada8

Data were imported into Excel and manually formatted such that: each sheet provides results of the screens begun on a single date. The plate reader OD600 data are presented in blocks of 8 rows by 12 columns, representing the layout of a 96 well plate. Each column of blocked data represents data from a time point, given in the first row of the sheet. Each row of blocked data represents one plate on one substrate, given in the first column of the sheet. 

Cells are conditionally colored based on value (blue low, red high), so that wells with growth defect can be readily observed. 

Mutant plate-well key
filename: Mutants_plate_well_key.xlsx

(Microsoft Excel, 16.52 KB)
MD5:5d396acf8e28183687f0718ad7c16898

Identifies the mutant strain in each well position of each plate. Columns indicate the plate number, well location in 96 well plate, locus tag of the disrupted gene, initial genome annotation of the gene, and the location within the gene of the transposon insertion. 

Column names and descriptions:
Plate: ID of arrayed mutant plate.
Well: the row and column location of the well in the 96 well plate.
Gene: locus tag of disrupted gene
Protein_previous_name: existing gene annotation at initiation of experiment, some annotations have changed.
Tn_Insertion_as_fraction_of_gene_length: location of transposon insertion within gene

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-2019589
Simons Foundation	542391

Deployments

None available yet

Instruments

plate reader

Supplied Name: SpectraMax M3 plate reader, Molecular Devices, San Jose, CA

Supplied Description:

Instrument Type

Generic Name: plate reader

Generic Description:

Plate readers (also known as microplate readers) are laboratory instruments designed to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 6-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well (8 by 12 matrix) with a typical reaction volume between 100 and 200 uL per well. Higher density microplates (384- or 1536-well microplates) are typically used for screening applications, when throughput (number of samples per day processed) and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization. From: http://en.wikipedia.org/wiki/Plate_reader, 2014-09-0-23.

Parameters

Date and time formats are described in python strftime() syntax.

Supplied Name	Supplied description	Supplied Units	Standard Name
date	date of experiment initiation in ISO Format Format: %Y-%m-%d	unitless	date
substrate	substrate screened as sole carbon source	unitless	sample_descrip
plate	ID of the arrayed mutant plate used	unitless	sample_descrip
time	time (hours) of sample	hours (hrs)	time_inc
well_row	row ID of well in 96 well plate	unitless	sample_descrip
well_col	column ID of well in 96 well plate	unitless	sample_descrip
OD	optical density read at 600 nm	nanometer (nm)	optical_density

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Database

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Dataset: High Throughput Growth Screen Data