Elaboration of an electroporation protocol for large plasmids and wild-type strains ofBacillus thuringiensis

To elaborate an effective electroporation protocol for large plasmids and wild type strains of Bacillus thuringiensis.The effect of DNA desalting, wall-weakening agency, cell growth conditions, electroporation solutions, and electric fields on electroporation efficiency was evaluated to optimize electroporation conditions for B. thuringiensis. By using this improved method, the greatest efficiency was reached 2 x 10(10 )CFU microg(-1) with pHT304, which is 10(4) times higher than previously reported. Four large plasmids (29.1, 44.9, 58 and 60 kb) were successfully transferred into the acrystalliferous B. thuringiensis strain BMB171; these results have not been achieved with previous protocols. Three wild type B. thuringiensis strains which could not be transformed previously were also transferred successfully.This improved method is more efficient for small plasmids; it is also appropriate for large plasmids and wild type B. thuringiensis strains which were not transformed by previous procedures.The present study established an effective electroporation protocol for large plasmids and wild type strains of B. thuringiensis. This method is well suited for the cloning and expression of huge DNA fragments such as gene clusters in B. thuringiensis. It also can be used as a reference method for other Bacillus strains that are refractory to electroporate.