Halomonas sp. O-1 is a halophilic bacterium with a high potential for industrial application due to its natural ability to produce polyhydroxyalkanoates (PHAs) using seawater-based media. However, a major barrier preventing industrial scale implementation of this organism is a lack of molecular methodologies capable of readily transforming members of the Halomonas genus. Currently, the only reliable method used for introducing DNA into Halomonas spp. is bacterial conjugation, a somewhat tedious and time-consuming technique compared to electroporation-based methodologies. Here we describe a rapid and reproducible method for the electroporation of Halomonas sp. O-1 with plasmid DNA. Electrocompetent cells were generated by growing Halomonas sp. O-1 in a yeast extract-tryptone medium with a final salinity of 3.5%, pH of 7.5, followed by several washes using 300mM sucrose. Results show that plasmids containing chloramphenicol (Cm(R)) and gentamicin (Gm(R)) resistance cassettes are suitable antibiotic selection markers for transformation and yields of 10(4) transformants per μg of DNA were obtained. This method is simple to perform and the materials used are readily available in most research labs. Additionally, this plasmid-based transformation procedure has the potential to be adapted for a number of applications including the creation of recombinant stains and the generation of deletion mutants of Halomonas spp.
Keywords: Competent cells; Electroporation; Halomonas; Transformation.
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