Journal of Microbiology

Effect of Temperature on Persistence of Recombinant Plasmid pCU103 in Different Waters: Kwak, Myong Ja , Kim, Chi Kyung , Kim, Young Chang , Lim, Jae Yun , Kim, Young Soo , Lee, Ki Sung , Min, Kyung Hee; J. Microbiol. 1995;33(3):178-183.

Abstract: The recombinant plasmid of pCU103 constructed by cloning pcbCD genes in pBluescript SK(+) was studied for the effect of temperature on its persistence in different waters by the methods of electrophoresis, Southern hybridization, quantification, and transformation. The plasmid was very rapidly degraded out in non-sterile FW water without regards to water temperature, probably due to the effect of biochemical factor such as nucleases. The pCU103 was most persistent at 4℃ in any water environments, moderately persistant at 15℃, but least stable at 30℃ such results could be explained by the facts that hydrogen bonds in double-stranded plasmid DNAs become unstable and that nucleases are activated by increasing temperature. The intact structure of pCU1-3 was generally observed by gel electrophoresis under the conditions which the plasmid should be 2.0 ng/㎕ or higher in concentration and that about 10² CFU/ml or more transformant cells should be recovered.

Structural and functional stability of the genetic recombinant plasmid pCU103 in different water environments: Kim, Chi Kyung , Kwak, Myoung Ja , Lee, Sung Gie; J. Microbiol. 1996;34(3):241-247.

Abstract: The stbility of the genetically engineered microorganisms and their recombinant plasmids released in natural environments has been regarded as one of the molecular ecological topics. In this study, the recombinant plasmids pCU103 in which the pcbCD genes involved in biodegradation of biphenyl and 4-chlorobiphenyl were cloned in pBluescript SK(+) vector, were examined for their structural and functional stability in different waters at 15℃ by the methods of electrophoresis, Southern hybridization, quantification with fluorescent dye, and transformation. The recombinant plamids maintained their stabilities for about 30 days in sterilized distilled water (SDW), 15 days in autoclaved creek water (AW), 25 days in filtered and autoclaved non-sterible creek water (FAW), 4 days in Luria-Bertani (LB) broth, and less than one day in filtered non-sterile creek water (FW). The covalently closed circular (CCC) form of the plasmid was decreased and open circular (OC) form was increased as a function of incubation time, and then linear (L) form was produced to be ultimately degraded out. The degradation rates of the plasmid were proportionally correlated to trophic level of the water, and the biological factor such as DNases was found to be one of the most critical factors affecting structural and functional stability of the plasmid in non-sterile natural water.

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