Optimization of Methods for Transfecting Spiroplasma citri Strain R8A2 HP with the Spiroplasma Virus SpV1 Replicative Form

• Published in: Plasmid Vol. 29; no. 3; pp. 193 - 205
• Main Authors: Gasparich, Gail E., Hackett, Kevin J., Stamburski, Corinne, Renaudin, Joël, Bové, Joseph M.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.05.1993; Elsevier
• Subjects: BACTERIOFAGOS; Bacteriological methods and techniques used in bacteriology; Bacteriology; BACTERIOPHAGE; Bacteriophages - genetics; Biological and medical sciences; Calcium Chloride - pharmacology; Chlorides - pharmacology; DNA, Viral - genetics; Evaluation Studies as Topic; Freezing; Fundamental and applied biological sciences. Psychology; GENETICA; GENETIQUE; Life Sciences; Liposomes; Microbiology; MYCOPLASMATALES; Polyethylene Glycols; Rubidium - pharmacology; Spiroplasma - genetics; Transfection; TRANSFORMACION GENETICA; TRANSFORMATION GENETIQUE; Performance evaluation; Genetic transformation; Optimization method; Spiroplasma citri; Electroporation; Replicative form; Experimental protocol; Mollicutes; Virus; Mycoplasmatales; Spiroplasmataceae; Transfection; Bacteria; Comparative study; VIROLOGIE; VIRUS SPV1
• ISSN: 0147-619X; 1095-9890
• DOI: 10.1006/plas.1993.1022

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 × 10-4 and 9.1 × 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.