Electroporation and its effect on the psychrotrophic bacterium Bacillus psychrophilus

Abstract

The psychrotrophic bacterium Bacillus psychrophilus was successfully transformed by electroporation with the cloning vector pC194 and the expression vector pPL708. Optimal electroporation parameters such as field strength, pulse length, and electroporation medium, as well as the influence of the growth phase of the bacterial cells, were determined. Maximum transformation efficiencies of 104 transformants/μg plasmid DNA were achieved with late logarithmic – early stationary phase cells grown in medium supplemented with 33.3 mM glycine and electroporated at high field strengths of 18–20 kV∙cm−1. By means of this procedure, a kanamycin-resistant gene was directly cloned into pPL708 and the recombinant plasmid electrotransformed into B. psychrophilus. In addition, the effect of electroporation on protein synthesis was analyzed by 2-dimensional polyacrylamide gel electrophoresis and computing scanning laser densitometry. At least eight proteins were induced by electroporation field strengths of 18 or 25 kV during the first 2 h immediately following electroporation. Conversely, the synthesis of at least five proteins was repressed by electroporation.Key words: electroporation, psychrotrophic bacterium.