Optimization of electroporation conditions for Arthrobacter simplex

Abstract

Arthrobacter simplex is an important industrial production strain with the steriod 1-en-dehydrogenation ability, which is widely applied in industry production [3]. However the genetic manipulation was obstructed because there was no efficient transformation protocol to date. In this study, we developed a detailed electroporation method through a systematic examination of the factors involved in the entire electroporation process, including the cell growth phase, wall-weakening treatment conditions, DNA amounts and the electroporation parameters. The optimized procedure was as follows: An inoculum of 1% from the first stage (exponential phase) was inoculated into fresh LB broth, and cultivated until the OD600 approached approximately 1.0. Penicillin was added at the final concentration of 70 μg/ml, and continued to cultivate for an additional 1 h. The mixture consisting of 60 μl of the competent cells and 200 ng of DNA was electrically transformed at a voltage of 22 kV/cm. After the electric pulse, the mixture was cultured in the recovery medium for 8 h. Finally, an efficiency of 3.76 × 104 transformants/μg DNA was obtained. This protocol was also successfully applied to other Arthrobacter species, such as Arthrobacter globisformis. Therefore, we conclude that the proposed method is rapid, simple and convenient, which allows a transformation trial to be accomplished in minutes.