DESIGN OF A NOVEL REGULATORY CIRCUIT FOR EXPRESSION OF RESTRICTION ENDONUCLEASES

We have developed a new strategy with a very tight control for the exp ression of cloned genes. The system employed here is the T7 promoter-b ased expression system in which transcription activator protein C of b acteriophage Mu (Mu C) has been cloned to serve as a repressor in the regulatory circuit. The system also includes pLysE, which encodes T7 l ysozyme, an inhibitor of T7 RNA polymerase. This ensures tight regulat ion of cloned genes in the uninduced state. Upon induction, the expres sed Mu C protein binds to its cognate site thereby repressing lys tran scription driven by the tet promoter. In order to evaluate the tight c ontrol achieved in the system, and to check leaky expression, if any, we have cloned the gene for the Smal restriction endonuclease without its cognate methylase. For this purpose, a dicistronic unit was constr ucted by cloning the smalR gene downstream of the Mu C gene. Smal expr ession was observed only in the induced cell extracts, demonstrating a tight control. The system could be used to express the genes of other cloned restriction enzymes and has the potential for general applicat ions.