Abstract
Plasmid DNAs in the range from 2.9 to 12.6 kbp were transferred into Bacillus subtilis ISW1214 intact cells by the use of electroporation. The transformation efficiency (transformants per μg plasmid DNA) decreased with increases of size of the DNA. However that of 2.0×103 transformants per μg of DNA were done routinely, by using a plasmid with a large molecular size of 12.6 kbp. The size of plasmid DNA in the range of 3.7 to 12.6 kbp did not affect the molecular efficiency (transformants per molecule input DNA). The transformation efficiency as high as 9.3×104 transformants per μg of purified plasmid pUB110 was obtained, using a cell concentration of 7.6×1010 cells/ml and DNA concentration of 4 μg/ml in buffer containing 0.3 M sucrose, 1 mM CaCl2, and 1 mM sodium citrate (pH 6.0) under optimal pulse conditions of an electric field strength of 7 kV/cm and a duration of 500 μs with a single squared pulse at 0°C. The gene expression for antibiotic resistance after electroporation was completed within 1.5 h. The transformants were confirmed to harbor the same intact plasmid by agarose gel electrophoretic analysis.
