4077

3855

كارشناسي ارشد

بيوتكنولوژي كشاورزي

اصفهان: دانشگاه صنعتي اصفهان: دانشكده كشاورزي

1386

هفده، 116، [II]ص.: مصور ، جدول، نمودار

ص. ع. به فارسي و انگليسي

رضا زارعي

حميدرضا رحماني

05/05/87

مسعود بهار، نفيسه نيلي

1396/03/02

كتابنامه

كشاورزي

مهندسي كشاورزي

ID3855

به فارسي و انگليسي: قابل رؤيت در نسخه ديجيتال

Abstract Polymerase Chain Reaction or PCR is a powerful tool for the amplification of selected regions of a DNA molecule The amplification reaction is particularly dependent on the activity of Polymerase enzymes that are heat resistant In fact only after the discovery and application of such polymerase enzymes PCR started to change into a prevalent and common technique in laboratories all over the world Polymerase enzymes not only differ in term of hear resistance but also in their error rates Pfu a DNA polymerase from Pyrococcus furiosus is one of these polymerases that combines a remarkable heat resistance with an integral 3 5 exonuclease activity proofreading resulting in one of the lowest error rates of any known polymerase used in PCR In this research optimum Pfu over expression in Escherichia coli under pET expression systems was investigated To this end three different constructs of Pfu coding sequences were prepared They were 1 unmodified Pfu consisting of a free standing Pfu gene in a pET 21c expression vector 2 Ubi Pfu consisting of a ubiquitin fusion partner upstream of a Pfu gene in a pET 21c expression vector and 3 pelB Pfu consisting of a pelB fusion partner upstream of a Pfu gene in a pET 22b Recombinant vectors were introduced into E coli strain BL21 that was subsequently grown in liquid TB and induced with IPTG Expression levels were compared by SDS PAGE It was found that expression of Ubi Pfu is similar to that of unmodified Pfu whereas pel Pfu expression was four to five folds more than the other two constructs Furthermore PCR reactions were used to assess the biological activity of the expressed enzymes It was determined that all three enzymes had the expected biological activity Comparing the PCR products of the three enzymes it was found that pel Pfu resulted in a considerably more biological activity compared to that from unmodified Pfu and Ubi Pfu an observation largely attributed to higher expression of pel Pfu as judged by SDS PAGE profiles explained abobe Since pelB is expected to direct recombinantly expressed proteins to the peripelasmic space presence of pel Pfu in this compartment was also investigated However neither o osmotic shocks nor thermal treatment at 55 C that are commonly used to extract periplasmic proteins in E coli were successfully used to this end Nonetheless this remained to be shown if pelB Pfu was not altogether secreted into the periplasmic space or it was secreted into the space but couldn t pass the external bacterial membrane due to its prohibitively large size

رضا زارعي

حميدرضا رحماني

مسعود بهار، نفيسه نيلي