Preliminary Study Of Recombining The Type â…¡s Enzymes

The type â…¡ restriction enzymes contain a domain recognizing and then binding with the DNA helixes and a catalyzing domain that cleaves it.Objective:Fokl and PI-SceI are two kinds of type â…¡ restriction enzymes. The catalyzing domain gene of FokI and the recognizing domain gene of PI-SceI were together cloned into the expression vector pET28a+, to prepare recombined vector for further expression of new fusion enzyme.Method:the study cloned these two gene fragments from Flavobacierium and Saccharomyces cerevisiae by PCR, and then attempted to clone them into the vector pET28a+.Result:the catalyzing domain gene of FokI is631bp, and the recognizing domain gene of PI-Scel is546bp. In the course of cloning, each gene fragment could be successfully cloned into the vector, and both could express a protein about20000Da that right to its size. But about600bp gene deletion was found by double digestion and PCR and sequencing analysis when the second fragment was cloned into the vector that combined the first gene fragment. Further examines were taken which shown that the fragments were safe in the presence of all the restriction enzymes used in the cloning. While the sequencing result of the recombined vector illustrated that the PI-SceI gene fragments was well cloned compare with the FokI gene fragments which was almost totally lost. Then the two gene fragments were connected together in vitro, and then examined by PCR. a250bp to500bp diffusion was found by the electrophoresis.Conclusion:The deletion may be caused by a cut owing to the host cell which rejected those two gene fragments in the conversion process due to their toxicity to the cell when these two gene fragments were connected together. The PCR analysis showed another case to the deletion that these two fragments could form into complex structures when connected together in vitro. There was a third image about the deletion that they might cut themselves when the two genes were linked together, but it was almost impossible.