Design of a Novel Regulatory Circuit for Expression of Restriction Endonucleases

Chandrashekharan, Siddamadappa and Paul, Bindu Diana and Nagaraja, Valakunja (1998) Design of a Novel Regulatory Circuit for Expression of Restriction Endonucleases. In: Biological Chemistry, 379 . pp. 579-582.

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Abstract

We have developed a new strategy with a very tight control for the expression of cloned genes. The system employed here is the T7 promoter-based expression system in which transcription activator protein C of bacteriophage Mu (Mu C) has been cloned to serve as a repressor in the regulatory circuit. The system also includes pLysE, which encodes T7 lysozyme, an inhibitor of T7 RNA polymerase. This ensures tight regulation of cloned genes in the uninduced state. Upon induction, the expressed Mu C protein binds to its cognate site thereby repressing lys transcription driven by the tet promoter. In order to evaluate the tight control 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 constructed by cloning the smalR gene downstream of the Mu C gene. Smal expression 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 applications.

Item Type:	Journal Article
Publication:	Biological Chemistry
Publisher:	Walter de Gruyter & Co
Additional Information:	Copyright for this article belongs to Walter de Gruyter & Co.
Keywords:	Artificial operon;Mu C protein;Smal endonuclease;T7 lysozyme;Transcription terminator;T7 RNA polymerase
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	09 Dec 2004
Last Modified:	19 Sep 2010 04:17
URI:	http://eprints.iisc.ac.in/id/eprint/2381

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