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Desulfovibrio vulgaris Hildenborough Genetic Constructs

Citation

Wall, Judy et al. (2021), Desulfovibrio vulgaris Hildenborough Genetic Constructs, Dryad, Dataset, https://doi.org/10.5061/dryad.h70rxwdh9

Abstract

The dissimilatory sulfate-reducing Deltaproteobacterium, Desulfovibrio vulgaris Hildenborough (ATCC 29579), was chosen by the LBNL-led research collaboration ENIGMA to explore tools and protocols for bringing this anaerobe to model status.  Here we describe a collection of genetic constructs generated by ENIGMA, which are available to the research community.

Methods

Standard molecular tools were used to construct plasmids that could be transformed into the D. vulgaris cells for selection of double homologous recombination to exchange a gene, operon or gene fragment with a selectable marker. These small plasmids were unable to replicate in this host and retention of the marker ensured insertion into the chromosome or native plasmid, pDV1. We discovered that double recombination events were almost 10-fold more frequent than single events.  Thus retention of the plasmid backbone occurred infrequently.  All PCR generated DNA fragments were sequenced before reintroduction into DvH for genetic constructs.

Plasmid construction:  Li and Elledge (2012;  DOI 10.1007/978-1-61779-564-0_5,) and Gibson Assembly (2009; DOI:10.1038/NMETH.1318).

Marker replacement plasmids:  Keller et al. (2011; DOI 10.1016/B978-0-12-385075-1.00022-6)

Markerless deletion plasmids: Moore and Leigh (2005; DOI: 10.1128/JB.187.3.972-979.2005) and Keller et al. (2009; DOI 10.1128/AEM.01839-09)

Transposon library: Larsen et al. (2002; DOI: 10.1007/s00203-002-0442-2).  Deutschbauer et al. (2011;DOI:10.1371/journal.pgen.1002385). Kuehl et al. (2014; DOI: 10.1128/mBio.01041-14). 

TAP-tagged protein production: Butland et al. (2005; DOI: 10.1038/nature03239).  Shatsky et al. (2016; DOI:10.1074/mcp.M115.054692)

Plasmids pSC27 (a shuttle vector with a native Deltaproteobacteria replicon), pMO9075 (for stable expression of genes for complementation studies in DvH), pMO719 (for marker exchange constructs in DvH), and pMO746 (for delivery of the gene for uracilphosphoribosyltransferase to DvH) are all available from Addgene, Inc.  These have the features used in our constructs.

Usage Notes

A ReadMe file is being uploaded which is associated with the lists of constructs and provides more detailed descriptions of and publications for the protocols used. 

Prior to pooling a specialized subset of transposon mutants to determine gene fitness data, all members should be streaked to determine the lack of aerobic contaminants.  A pool of the ca. 11,000 mutants isolated and sequenced was found to have contamination.  Of the mutants we have examined, only one or two showed contamination.  Often a second transposon mutant interrupting the same gene (usually a different site) is part of the collection that may show no contamination.

Select publications from this work are noted in the ReadMe file and in the Excel file "Genetic Constructs of Desulfovibrio vulgaris Hildenborough final" in sheet 5, "Boutique Deletions," column F.

 

Funding

U.S. Department of Energy, Award: DE-AC02-05CH11231

University of Missouri, Award: Mizzou Advantage to KD

U.S. Department of Energy, Award: DE-FG02-08ER64691