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:: Volume 9, Issue 1 (8-2020) ::
2020, 9(1): 11-18 Back to browse issues page
Establishment of molybdeum cofactor detection system in Escherichia coli
Kiana Kabir, Khalil Zaynali Nezhad , Andreas Weber, Seyede Sanaz Ramezanpour, Marion Eisenhut
Department of Plant Breeding and Biotechnology, College of Plant Production, Gorgan University of Agricultural Science and Natural Resources, Iran , Khalil1381@yahoo.com
Abstract:   (1288 Views)
Biotin sulfoxide reductase enzyme is one of the enzymes involved in adaptation to different environmental conditions. BisC enzyme converts biotin sulfoxide to biotin and Methionine sulfoxide to Methionine under oxidative stress conditions. Both free Methionine and protein bound chains Methionine are essential in protein synthesis process and the function of proteins. The activity of the BisC enzyme is dependent to the Bis-MGD as a co-factor. In the current study, in order to verify the presence of bacterial Molybdeum cofactor, an indirect approach was made by showing the activity of BisC enzyme as a reporter gene. Therefore, in this regard BisC gene was cloned to pJet vector and was transferred to Mach1 strain of E. coli by heat shock transformation. The clones were selected on LB medium complemented with 200 µg/ml of Ampicillin antibiotic and the PCR reaction was done. After verifying of positive clones by sequencing, the BisC gene was digested and ligated to the pETDuet expression vector and transferred to Rossetta. Over-expression of BisC enzyme was analyzed on a SDS-PAGE gel and the activity of the enzyme could be showed as a colorless band on the Native gel by oxidation of methyl viologen. It proved the presence of active Bis-MGD as a cofactor.
Keywords: Key words: Bis-MGD, Biotin sulfoxide, Escherichia coli, Molybdenum, Over expression
Full-Text [PDF 733 kb]   (350 Downloads)    
Type of Study: Research | Subject: Plant
Received: 2020/04/16 | Accepted: 2020/09/16 | Published: 2020/09/16
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Kabir K, Zaynali Nezhad K, Weber A, Ramezanpour S S, Eisenhut M. Establishment of molybdeum cofactor detection system in Escherichia coli. Genetic Engineering and Biosafety Journal. 2020; 9 (1) :11-18
URL: http://gebsj.ir/article-1-342-en.html


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Volume 9, Issue 1 (8-2020) Back to browse issues page
دوفصل نامه علمی-پژوهشی مهندسی ژنتیک و ایمنی زیستی Genetic Engineering and Biosafety Journal
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