Desulfovibrio vulgaris Hildenborough Genetic Constructs

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.