Genetic transformation of bacillus subtilis with liposome-entrapped plasmid pUB110

Abstract

Chromosomal DNA fragments as well as pUB110 plasmid DNA were entrapped in liposomes and transformation of $\underline{Bacillus}$ $\underline{subtilis}$ protoplasts with the entrapped DNA was tried for the purpose of increasing transformation efficiency. $\underline{B}$.$\underline{subtilis}$ chromosomal DNA fragments of 10-15 megadalton sizes were encapsulated in large unilamellar vesicles (LUV) using ether injection method. The same injection method was used for encapsulating plasmid pUB110 into the LUV. The liposome containing DNA were then tried to fuse with $\underline{B}.$\underline{subtilis}$ protoplasts under the presence of polyethyleneglycol (PEG) as usual. The formation of LUV having 0.1μm average diameter was confirmed by election microscopy. The average weight of the LUV was found to be more than 2 megadaltons. The $^3H$-labeled chromosomal DNA of $\underline{B}$.$\underline{subtilis}$ was entrapped in liposomes and the liposome suspension was applied to Sepharose CL-4B column. The entrapment of chromosomal DNA in the liposomes was proved by coincident peaks of radioactivity and optical density at fraction number 15. About 5μl of chromosomal DNA was entrapped in 1μmol of liposome. Intact cells of the two tryptophan requiring mutants of $\underline{B}$.$\underline{subtilis}$ 168 and BD170, were found to mutate reversely to non-requiring strains with frequency in the order of $10^{-6}$. This frequency, however, increased by 100-fold when the cells were protoplasted. The mechanism involved is remained to be studied. Entrapment of plasmid pUB110 DNA into liposome was also confirmed by the agarose gel electrophoresis. The transformation of $\underline{B}$.$\underline{subtilis}$ protoplasts with liposome-entrapped plasmid DNA was succeeded, but the frequency was about 10-fold lower than that of PEG-induced protoplast transformation.