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Molecular Analysis of the Clavulanic Acid Regulatory Gene Isolated from an Iranian Strain of Streptomyces Clavuligerus , PTCC 1709

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 st...

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Autores principales: Hojati, Zohreh, Salehi, Zahra, Motovali-Bashi, Majid, Korbekandi, Hasan, Jami, Saeed
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Royan Institute 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584469/
https://www.ncbi.nlm.nih.gov/pubmed/23508694
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author Hojati, Zohreh
Salehi, Zahra
Motovali-Bashi, Majid
Korbekandi, Hasan
Jami, Saeed
author_facet Hojati, Zohreh
Salehi, Zahra
Motovali-Bashi, Majid
Korbekandi, Hasan
Jami, Saeed
author_sort Hojati, Zohreh
collection PubMed
description 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 subjected 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 isolated from S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymorphism (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 overproduced protein could also be used for other related investigations, such as a mobility shift assay.
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spelling pubmed-35844692013-03-18 Molecular Analysis of the Clavulanic Acid Regulatory Gene Isolated from an Iranian Strain of Streptomyces Clavuligerus , PTCC 1709 Hojati, Zohreh Salehi, Zahra Motovali-Bashi, Majid Korbekandi, Hasan Jami, Saeed Cell J Research Article 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 subjected 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 isolated from S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymorphism (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 overproduced protein could also be used for other related investigations, such as a mobility shift assay. Royan Institute 2011 2011-09-23 /pmc/articles/PMC3584469/ /pubmed/23508694 Text en Any use, distribution, reproduction or abstract of this publication in any medium, with the exception of commercial purposes, is permitted provided the original work is properly cited http://creativecommons.org/licenses/by/2.5/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Hojati, Zohreh
Salehi, Zahra
Motovali-Bashi, Majid
Korbekandi, Hasan
Jami, Saeed
Molecular Analysis of the Clavulanic Acid Regulatory Gene Isolated from an Iranian Strain of Streptomyces Clavuligerus , PTCC 1709
title Molecular Analysis of the Clavulanic Acid Regulatory Gene Isolated from an Iranian Strain of Streptomyces Clavuligerus , PTCC 1709
title_full Molecular Analysis of the Clavulanic Acid Regulatory Gene Isolated from an Iranian Strain of Streptomyces Clavuligerus , PTCC 1709
title_fullStr Molecular Analysis of the Clavulanic Acid Regulatory Gene Isolated from an Iranian Strain of Streptomyces Clavuligerus , PTCC 1709
title_full_unstemmed Molecular Analysis of the Clavulanic Acid Regulatory Gene Isolated from an Iranian Strain of Streptomyces Clavuligerus , PTCC 1709
title_short Molecular Analysis of the Clavulanic Acid Regulatory Gene Isolated from an Iranian Strain of Streptomyces Clavuligerus , PTCC 1709
title_sort molecular analysis of the clavulanic acid regulatory gene isolated from an iranian strain of streptomyces clavuligerus , ptcc 1709
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584469/
https://www.ncbi.nlm.nih.gov/pubmed/23508694
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