Development of Eukaryotic (yeast) Expression System for Promoters Characterization and Recombinant Proteins Production

Abstract

Promoter is a Crucial genetic element that plays major role in the expression of gens expression. It is a part of DNA located upstream of gene where relative enzymes bind to start transcription. Its structure, stability, and affinity with RNA polymerase influence the gene expression. The main focus of this research is UBC9_1 promoter of Saccharomyces cerevisiae, a unicellular eukaryotic model organism due to its known genetics and completely characterized physiology. Its promoters are widely characterized due to biotechnological usefulness because of fermentation capacity and production of bioethanol and other useful compounds. This study aims to check the expression of the EGFP reporter gene under the UBC9_1 promoter via yeast expression vector pCEV-G4-km to access its rate of expression with the control CYC1 promoter. Expression cassette designed, promoter and gene construct were synthesized and ligated into expression vector before CYC1 terminator, then transformed into E. coli BL21 to check the expression of the EGFP gene. Successful ligation of the construct into the vector and transformation is confirmed by the restriction digestion and colony PCR. After confirmation of transformation samples were sent for flow cytometry. Comparison of Side Scatted lin and Forward Scattered lin on Scatter plot of flow cytometry shows the greater expression of EGFP under UBC9_1 promoter as compared to control CYC1. Furthermore, analysis of the Florescence channel count shows greater Activity of EGFP expression under the UBC9_1 promoter. The geometric mean values of the UBC9_1 promoter are compared with the Control CYC1 promoter which also indicates a higher rate of expression of the UBC9_1 promoter.