title: Optimization of methods for transfecting Spiroplasma citri strain R8A2 HP with the spiroplasma virus SpV1 replicative form.
abstract: Seven methods for the transfection of bacteria were compared and optimized for use in Spiroplasma citri strain HP using the spiroplasma virus SpV1 R8A2 B replicative form (RF). These methods included both chemical-mediated protocols [CaCl2, RbCl/CaCl2, polyethylene glycol (PEG)], liposome-mediated transfection, electroporation, freeze/thaw cycling, and natural competence. The best protocols were those which utilized PEG or electroporation, yielding transfection frequencies of 1.4 x 10(-4) and 9.1 x 10(-4) transfectants/colony-forming unit (CFU), respectively. For both of these protocols, transfection frequencies were higher using CsCl-purified, covalently closed, circular DNA. In the PEG-mediated protocol, Sigma 8000 brand PEG at a final concentration of 44%, and the presence of carrier DNA proved to be optimal with a PEG exposure time of 2 min. Using electroporation, a 1-2 ms pulse of a 6.5 kV/cm electric field was best; washing the host cell pellet prior to electroporation enhanced efficiencies by 50%. Linearization of the DNA resulted in lower transfection efficiencies by either method.
authors.affiliation: Insect Biocontrol Laboratory, USDA, Beltsville, Maryland 20705.
authors.forename: G E
authors.initials: GE
authors.lastname: Gasparich
authors.forename: K J
authors.initials: KJ
authors.lastname: Hackett
authors.forename: C
authors.initials: C
authors.lastname: Stamburski
authors.forename: J
authors.initials: J
authors.lastname: Renaudin
authors.forename: J M
authors.initials: JM
authors.lastname: Bové
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D002122:Calcium Chloride / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004279:DNA, Viral / Q000235:genetics*; D005069:Evaluation Studies as Topic; D005615:Freezing; D008081:Liposomes; D011092:Polyethylene Glycols; D012413:Rubidium / Q000494:pharmacology; D013149:Spiroplasma / Q000235:genetics*; D014162:Transfection
chemical_list: D002712:Chlorides; D004279:DNA, Viral; D008081:Liposomes; D011092:Polyethylene Glycols; D002122:Calcium Chloride; D012413:Rubidium; C032710:rubidium chloride