Genetic transformation of bacilli protoplasts with plasmid pUB 110

Abstract

Plasmid pUB110 which was originated from $\underline{Staphylococcus aureus}$ and has been known to be expressed and maintained in $\underline{Bacillus subtilis}$ was tried to transfer into protoplasts of $\underline{B}$. $\underline{cereus}$ for the purpose of confirming possibility of utilizing the plasmid as a shuttle vector between the two species. About 60% of transformation was obtained by adjusting the amount of plasmid DNA to be used as 1g and the time for PEG treatment as 2 minutes. About 90 minutes of incubation was needed before transferred to the regeneration medium for the transformed protoplasts to regenerate with highest efficiency on the regeneration medium. The presence of recombination and restriction-modification system in $\underline{B}$. $\underline{subtilis}$ host cells did not affect on the acceptance of the plasmid DNA into the protoplasts. Fresh cells of $\underline{B}$. $\underline{cereus}$ grown in B-broth were harvested at the late log phase and protoplasted. The naked protoplasts were then transformed with pUB110 plasmid DNA. The successful transformation was confirmed by observing plasmid DNA band in the electrophoresis patterns of the transformant cell lysates. Further confirmation was made by the kanamycin resistance in the transformed $\underline{B}$. $\underline{cereus}$ cells which had been sensitive to the antibiotic before transformed.