Author/Authors :
Hojati، Zohreh نويسنده , , Salehi، Zahra نويسنده Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran , , Motovali-Bashi، Majid نويسنده , , Korbekandi، Hasan نويسنده Department of Genetics and Molecular Biology, Faculty of Medicine, Medical University of Isfahan, Isfahan, Iran , , Jami، Saeed نويسنده Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran ,
Abstract :
Objective: The clavulanic acid regulatory gene (claR) is in the clavulanic acid biosynthetic
gene cluster that encodes ClaR. This protein is a putative regulator of the late steps of clavulanic acid biosynthesis. The aim of this research is the molecular cloning of claR, isolated from the Iranian strain of Streptomyces clavuligerus (S. clavuligerus).
Materials and Methods: In this experimental study, two different strains of S. clavuligerus were used (PTCC 1705 and DSM 738), of which there is no claR sequence record for strain PTCC 1705 in all three main gene banks. The specific designed primers were sub- jected to a few base modifications for introduction of the recognition sites of BamHI and ClaI. The claR gene was amplified by polymerase chain reaction (PCR) using DNA iso- lated from S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymor- phism (PCR-RFLP), and sequencing were used for molecular analysis of the claR gene. The confirmed claR was subjected to double digestion with BamHI and ClaI. The cut claR was ligated into a pBluescript (pBs) vector and transformed into E. coli.
Results: The entire sequence of the isolated claR (Iranian strain) was identified. The presence of the recombinant vector in the transformed colonies was confirmed by the colony-PCR procedure. The correct structure of the recombinant vector, isolated from the transformed E. coli, was confirmed using gel electrophoresis, PCR, and double digestion with restriction enzymes.
Conclusion: The constructed recombinant cassette, named pZSclaR, can be regarded as an appropriate tool for site directed mutagenesis and sub-cloning. At this time, claR has been cloned accompanied with its precisely selected promoter so it could be used in expression vectors. Hence the ClaR is known as a putative regulatory protein. The over- produced protein could also be used for other related investigations, such as a mobility shift assay.
