Construction of GFP gene into yeast vector

Nur Fashareena Adila, Binti Abdul Rahim. (2014) Construction of GFP gene into yeast vector. [Final Year Project Report] (Unpublished)

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Abstract

Green Fluorescence Protein (GFP) originally isolated from Jellyfi sh~ Aequorea Vic/ona. The features of nontoxic and stable has make GFP become one of the favorable reporter genes. GFP is widely used for the purpose of monitoring the gene expression and selecting transgenic cells. pPICZa vector acquire important componen ts like, AOX 1 promoter, Zeocin resistance gene and alpha-fac tor signal sequence. These components allow the expression and secretion of recombinant protein in the methanogemc yeast Pichia pasfor/s. Pichia pastons 1S one of the main expression systems in yeast and acquired added feahJres hke easy maintenance and higher expression level. The main objective of this study is to clone the GFP gene IOta the pP leZaA. vector and to deterrnme the successful of the cloning by USll1g colony peR. For the constructlOn purpose, pAGS/GFP gene was amplified by using PCR protocol. Then, the PCR product was ligated into pGEM-T vec tor men sub clone into pPICZaA. expression vector. The alternative way 111 sub-clone the GFP gene direc tl y into pP ICZaA vector was also conducted via Restric tion Enzyme (RE) digestion me thod. The presence of positive transfonnant that contamed pPICZaAIGFP was confirmed by using colony peR sc reening. The construction of pPICZaA. IGFP that was obtained from this shJd y can be used fo r future researc h on the expression of recombinant GFP in Pichia pas/oris expression host.

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