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.
