Design of a novel regulatory circuit for expression of restriction endonucleases.
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 SmaI restriction endonuclease without its cognate methylase. For this purpose, a dicistronic unit was constructed by cloning the smaIR gene downstream of the Mu C gene. SmaI 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.
Chandrashekharan
S
S
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore.
Paul
B D
BD
Nagaraja
V
V
D017123:Bacteriophage T7 / Q000235:genetics; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D014764:Viral Proteins
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C090591:C protein, Bacteriophage Mu; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014764:Viral Proteins; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific