REMOVAL OF THE PERIPLASMIC DNASE BEFORE ELECTROPORATION ENHANCES EFFICIENCY OF TRANSFORMATION IN THE MARINE BACTERIUM VIBRIO-ALGINOLYTICUS

We have established a reliable procedure for electroporation in the ma rine bacterium Vibrio alginolyticus. Plasmids carrying the P15A replic on were found to be stably maintained in the Vibrio cells, and chloram phenicol, kanamycin or tetracycline were used for selection. Since we found that the Vibrio cells excrete DNase into the culture medium, cel ls were subjected to osmotic shock before extensive washing in order t o remove the DNase from the periplasmic space. This manipulation resul ted in about a 10-fold increase in the efficiency of transformation. I n 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 pha se, and when electroporation was carried out at an electric field stre ngth between 5.0 and 7.5 kV cm(-1). Under optimal conditions, about 10 (5) transformants per mu g of input DNA were reproducibly obtained, wh ich is tolerable for cloning.