EFFECTS OF PLASMID DNA SIZES AND SEVERAL OTHER FACTORS ON TRANSFORMATION OF BACILLUS-SUBTILIS ISW1214 WITH PLASMID DNA BY ELECTROPORATION

Plasmid DNAs in the range from 2.9 to 12.6 kbp were transferred into B acillus subtilis ISW1214 intact cells by the use of electroporation, T he transformation efficiency (transformants per pg plasmid DNA) decrea sed with increases of size of the DNA, However that of 2.0 x 10(3) tra nsformants per pg 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 ran ge of 3.7 to 12.6 kbp did not affect the molecular efficiency (transfo rmants per molecule input DNA), The transformation efficiency as high as 9.3 x 10(4) transformants per mu g of purified plasmid pUB110 was o btained, using a cell concentration of 7.6 x 10(10) cells/ml and DNA c oncentration of 4 mu g/ml in buffer containing 0.3 M sucrose, 1 mM CaC l2, and 1mM sodium citrate (pH 6.0) under optimal pulse conditions of an electric field strength of 7 kV/cm and a duration of 500 mu s with a single squared pulse at 0 degrees C. The gene expression for antibio tic resistance after electroporation was completed within 1.5 h. The t ransformants were confirmed to harbor the same intact plasmid by agaro se gel electrophoretic analysis.