Effects of Plasmid DNA Sizes and Several Other Factors on Transformation of Bacillus subtilis ISW1214 with Plasmid DNA by Electroporation

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 1995, Vol.59 (8), p.1433-1437
• Main Authors: Ohse, Morimasa, Takahashi, Kuniharu, Kadowaki, Yasuhiro, Kusaoke, Hideo
• Format: Article
• Language: English
• Subjects: Bacillus subtilis; Bacillus subtilis - genetics; Biological and medical sciences; Biotechnology; Electroporation; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Methods. Procedures. Technologies; Particle Size; Plasmids; Stem Cells; Time Factors; Transformation, Bacterial; Vectors (cloning, transfer, expression). Insertion sequences and transposons
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.59.1433

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 (transform ants per μg plasmid DNA) decreased with increases of size of the DNA. However that of 2.0 x 10 3 transform ants 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 (transform ants per molecule input DNA). The transformation efficiency as high as 9.3 x 10 4 transformants per μg of purified plasmid pUBII0 was obtained, using a cell concentration of 7.6 x 10 10 cells/ml and DNA concentration of 4μg/ml in buffer containing 0.3 M sucrose, 1 mM CaCl 2 , 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 500μs 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.