The Method And Mechanism Research On Ultrasound-mediated Transfer Of Plasmid PUC19 Into Escherichia Coli Competent Cells

Biological transformation was referred to as the phenomenon that the host strain accepted the gene from the donor strain, integrating into their own genomes, then obtained new genetic character. At present, with the development of transformation,some methods such as chemical transformation, electro-transformation,microprojectile bombardment transformation and ultrasound transformation have been widely used in the biology experiment. There has been no systematic reports on transformation mechanism, which was related to not only the character of exogenous DNA, but also the character of host strain.In this study, plasmid p UC19 was transferred into Escherichia coli DH5α by ultrasound. Moreover, to reveal the mechanism of transformation, we established the model of ultrasound mediated exogenous DNA into the host cells. The difference of the ingredients of the cell wall, cell membrane permeability, outer membrane protein genes of Escherichia coli DH5α before and after treatment by ultrasound were investigated, respectively. Besides, the mrd B gene mutations was constructed which existed in outer membrane protein.The results showed that ultrasound treatment could mediate exogenous DNA into competent cells in the presence of divalent metal ions. The transformation efficiency could reach at 1.006×107 CFU/μg DNA when the ultrasonic conditions were 35℃,40 W, 25 s and OD600=0.4⁰.6. The changed cell membrane permeability of E. coli DH5α was found that the change of cell membrane permeability treated by ultrasound is 10 times than that without ultrasound treatment under the optimal conditions. The transformation was out of proportion to the host cells survival rate, indicating that the transformation process was the result of combined action of exogenous DNA and host cells, because ultrasonic wave has a certain damage not only to the host cells, but also to the integrity of the exogenous DNA. The results of interactions among petidoglycan, metal ions and DNA showed that: Adsorption for metal ions of petidogly have no specificity, and it can react with both univalent metal ions（Na+）and bivalent metal ions(Mg²⁺, Ca²⁺), which had no relation to DNA, manifesting that a part of bivalent metal ions can offer ions channel for exogenous DNA entering host cells during the transformation after interaction with petidogly, but can not explain that the existence of petidoglycan play a dominant role in the transformation process.According to the result of bioactive macromolecular substances detection and electron microscopy observation, it showed that the cavitation caused by ultrasound changed the permeability of cell membrane and the structure of cell wall, also caused the surface of E. coli DH5α appear fold and unequal shape, which led to the bioactive macromolecular substances effuse out of cells through the cell membrane and also provided the foreign power for exogenous DNA entering into host cells. This research the mrd B gene mutations was also builded which existed in outer membrane protein,however, with a regret that the function on growth and transformation of this mutations needs further studying.We drew the following conclusions based on all the above results: The changed membrane permeability of host cells is the basic condition for exogenous DNAentering into the host strain. That bivalent metal ions interact with peptidoglycan can offer ions channel for exogenous DNA entering into host cells during the transformation. Ultrasonic treatment may cause macromolecular substances of the host cell effuse out of cells, which also offer ions channel and basic conditions for exogenous DNA entering into host cells. In this paper, some basic conditions were speculated based on physiological characteristics of E. coli DH5α, the mrd B gene mutations was also builded which existed in outer membrane protein. We believed that with the in-depth study of molecular biology, the mechanism of transformation would gradually be discovered to better service for scientific research and production.