An improved Eschehchia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation |
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Authors: | Z Shao WA Dick RM Behki |
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Institution: | Centre for Land and Biological Resources Research, Research Branch, Agriculture Canada, Ottawa, Ontario, Canada;School of Natural Resources, Ohio State University, Wooster, OH, USA |
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Abstract: | The genetic studies of metabolically diverse Rhodococcus spp. have been hampered by the lack of a system of introducing exogenous DNA. The authors improved an existing Escherichia coli-Rhodococcus shuttle vector (pMVS301) by removing much of the DNA not needed for replication and adding a multicloning site. This improved vector (pBS305) is 7·9 kb in length. Its ability to transform Rhodococcus was tested using electroporation parameters optimized for introduction of pMVS301 into Rhodococcus. Transformation efficiencies as high as 105 cfu μg-1 DNA were obtained although efficiencies varied depending on the Rhodococcus strain tested. The improved vector pBS305 offers great utility for genetic studies of Rhodococcus because its small size enables movement of large inserts of DNA into Rhodococcus , it has multicloning sites, contains a highly selective thiostrepton marker, and can be replicated in both E. coli and Rhodococcus. |
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