Effects of DNA topology on transformation efficiency of Bacillus subtilis ISW1214 by electroporation.
We report an investigation of electrotransformation by three different topological isomers, circular supercoiled (sc DNA), circular relaxed (cr DNA), and linearized (In DNA) forms of the plasmids pUB110 (4.5 kbp) and pBDR331T (12.6 kbp), of a Gram-positive bacterium, Bacillus subtilis ISW1214. Treatment of the sc DNA with calf thymus topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of sc DNA with restriction endonculease linearized the DNA. The transformation with the sc DNA of pUB110 resulted in the maximum efficiency of (2.6 +/- 0.6) x 10(5) transformants per microgram DNA higher than that (2.0 +/- 0.3) x 10(4) transformants per microgram DNA for the cr DNA, using the DNA concentration of 20 micrograms/ml at an electric field strength of 7 kV/cm and a capacitance of 10 microF with a single decayed pulse. The transformation efficiency (TE) for the ln DNA was zero. The variations of TE for different topological forms of DNA reflected their relative stability in the host cells. The molecular efficiency (ME, transformants per molecule) for sc DNA was nearly one order of magnitude greater for the lower molecular size of pUB110 DNA than that for the higher molecular size of pBDR331T DNA.
Ohse
M
M
Department of Applied Physics and Chemistry, Faculty of Engineering, Fukui University of Technology, Japan.
Kawade
K
K
Kusaoke
H
H
D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins; D004587:Electrophoresis, Agar Gel; D018274:Electroporation; D007536:Isomerism; D008970:Molecular Weight; D010957:Plasmids / Q000378:metabolism*; D015183:Restriction Mapping; D013950:Thymus Gland / Q000201:enzymology; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D004270:DNA, Circular; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D004264:DNA Topoisomerases, Type I