Removal of the periplasmic DNase before electroporation enhances efficiency of transformation in the marine bacterium Vibrio alginolyticus

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Abstract
We have established a reliable procedure for electroporation in the marine bacterium Vibrio alginolyticus. Plasmids carrying the P15A replicon were found to be stably maintained in the Vibrio cells, and chloramphenicol, kanamycin or tetracycline were used for selection. Since we found that the Vibrio cells excrete DNase into the culture medium, cells were subjected to osmotic shock before extensive washing in order to remove the DNase from the periplasmic space. This manipulation resulted in about a 10-fold increase in the efficiency of transformation. In addition, cells were washed in the presence of 5-10 mM Mg2+in order to stabilize the outer membrane. The efficiency of transformation was found to be optimal when cells were harvested at early stationary phase, and when electroporation was carried out at an electric field strength between 5-0 and 7.5 kV cm-1. Under optimal conditions, about 105transformants per μg of input DNA were reproducibly obtained, which is tolerable for cloning.

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