The Cloning And The Assay Of The Activity Of ColE2 Plasmid Replication Enhancer

It is reported by Itoh lab that there exists a DNA fragment downstream of ColE2 origin which can enhance the replication of ColE2 DNA and hence is named as enhancer II. And, they hypothesized that enhancer II can enhance the binding between ColE2 origin and Rep protein or make the Rep protein easier to be replaced by DNA polymerase I after the primer RNA is synthesized. In order to study the mechanism of the function of enhancer II, the recombinant plasmid pBluescript-ColE2 ori-enhancer II was constructed. Firstly, a pair of PCR primers with Xho I and EcoR I at either end were designed according to the sequence ColE2 origin and enhancer II. Next, the ColE2 ori-enhancer II was amplified from pEC22 plasmid. After being digested by Xho I and EcoR I, the ColE2 ori-enhancer II was ligated to pBluescript II SK+. After restriction digestion and gel analysis and PCR analysis, the positive recombinants were selected. Then, the sequence of the selected positive recombinants were confirmed by DNA sequencing, and the recombinant plasmid pBluescript-ColE2 ori-enhancer II was obtained. Finally, the pBluescript-ColE2 ori-enhancer II was used to measure the Rep binding activity of ColE2 ori-enhancer II. The results suggested that enhancer II functions because it can enhance the binding of ColE2 origin and Rep protein.