Efficient DNA transformation of Bradyrhizobium japonicum by electroporation.
Intact cells of Bradyrhizobium japonicum USDA 110 were transformed with a 30-kilobase plasmid to efficiencies of 10(6) to 10(7) transformants per microgram by high-voltage electroporation. The technique was reliable and simple, with single colonies arising from transformed cells within 5 days of antibiotic selection. Plasmid DNA from B. japonicum transformed the Bradyrhizobium (Arachis) sp. with high efficiency, while the same plasmid extracted from Escherichia coli transformed B. japonicum at very low efficiency. The electrical conditions that resulted in the highest efficiencies were high voltage (10.5 to 12.5 kV/cm) and short pulse length (6 to 7 ms). A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude; saturation appeared to begin between 120 ng/ml and 1.2 micrograms/ml. This novel method of transformation should enhance B. japonicum genetic research by providing a valuable alternative to conjugal mating, which is currently the only efficient, widely used means of introducing DNA into this organism.
Hattermann
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DR
Department of Microbiology, University of Tennessee, Knoxville 37996-0845.
Stacey
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D004269:DNA, Bacterial / Q000235:genetics*; D004560:Electricity; D005821:Genetic Techniques; D010957:Plasmids; D012230:Rhizobiaceae / Q000235:genetics*; D014170:Transformation, Genetic
D004269:DNA, Bacterial