Protocol for the transformation of Bacillus subtilis through inducing natural competence

Abstract

Transformation is the key step in genetic engineering as it governs the facile introduction of plasmid DNA or genome editing tools into the microbial cells. More importantly, approach taken for transformation differs between different microbial chassis depending on the structure of the cell envelope and other barriers. In this aspect, Bacillus subtilis requires a different transformation technique compared to the heat shock approach commonly used for Escherichia coli. Given that B. subtilis enters competence state during stationary phase, DNA could theoretically be introduced into the Gram-positive bacterium during this differentiation program. This aspect of B. subtilis cell biology has been exploited in biotechnology through the use of xylose for inducing the comK master competence regulator. With xylose as inducer, comK gene is transcribed, which would set off a transcriptional program that guides B. subtilis stationary phase cells to enter the natural competence state, where the cell envelope of the bacterium is permeabilized to afford facile entry of plasmid DNA. This protocol details the experimental approach and procedure for inducing natural competence in B. subtilis cells for enabling plasmid DNA to enter the bacterial cell during transformation. One methodological innovation in the described protocol is the use of stationary phase B. subtilis cells as compared to exponential phase cells in earlier incarnations of the approach.