Optimization of the spizizen method for wild-type Bacillus subtilis N4 transformation

The constructed large recombinant plasmid could not be transformed into the wild-type Bacillus subtilis N4 by traditional spizizen method. In this study, we optimized the spizizen method by adding organic solvents and lysozyme in preparation and transformation of competent cells. The types and concentrations of organic solvent, the concentrations of lysozyme, co- culture time of plasmids and competent cells were studied for improving transformation efficiency to construct Bacillus subtilis N4 PHT01-nit. After IPTG induced, the expression of nit in wild type Bacillus subtilis N4 was validated by SDS-PAGE.Results showed that the highest transformation efficiency of 37 transformants· μg- 1DNA could be obtained when 4% of the Tween-80 was added in preparation of competent cells, 5 μg· m L-1of lysozyme was added in transformation of competent cells, and plasmids were cultured with competent cells for 1 h. SDS- PAGE showed that the target protein band was consistent with nitrilase. This optimized spizizen method could be used for large plasmid DNA transformation into the wild-type Bacillus subtilis.