1887

Abstract

We have established a reliable procedure for electroporation in the marine bacterium . Plasmids carrying the P15A replicon were found to be stably maintained in the cells, and chloramphenicol, kanamycin or tetracycline were used for selection. Since we found that the 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 Mgin 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. Under optimal conditions, about 10transformants per μg of input DNA were reproducibly obtained, which is tolerable for cloning.

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1994-09-01
2021-07-29
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