title: A rapid and efficient electroporation method for transformation of Halomonas sp. O-1.
abstract: 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.
authors.affiliation: Department of Chemistry, SUNY College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, USA.
authors.forename: Joshua R
authors.initials: JR
authors.lastname: Harris
authors.affiliation: Department of Chemistry, SUNY College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, USA.
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Lundgren
authors.affiliation: Department of Chemistry, SUNY College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, USA.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Grzeskowiak
authors.affiliation: Department of Creative Engineering, National Institute of Technology, Kitakyushu College, 5-20-1 Shii, Kokuraminami-ku, Kitakyushu 802-0985, Japan.
authors.forename: Kouhei
authors.initials: K
authors.lastname: Mizuno
authors.affiliation: Department of Chemistry, SUNY College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, USA; Center for Applied Microbiology, SUNY College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, USA; Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, College of Life Sciences, Hubei University, Hubei Province, People's Republic of China. Electronic address: ctnomura@esf.edu.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Nomura
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D020632:Halomonas / Q000235:genetics*; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D054712:Salinity; D012623:Seawater / Q000382:microbiology; D014169:Transformation, Bacterial
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Competent cells; Electroporation; Halomonas; Transformation