KONSTRUKSI PLASMID REKOMBINAN UNTUK EKSPRESI GEN AMORFA-4,11-DIEN SINTASE (ADS) ARTEMISIA ANNUA L. PADA SACCHAROMYCES CEREVISIAE BY4741

• Main Author: St. Tamara Chrysanthy, Nd.
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/40381
• Institution: Institut Teknologi Bandung

Malaria is a disease caused by a parasite, Plasmodium sp. According to WHO recommendation, Artemisinin-based Combination Therapy (ACT) can be used as treatment for malaria, which contain artemisinin or its derivate combining with other antimalarial drugs. Artemisinin is a secondary metabolite in Artemisia annua L. that is naturally produced in low concentration. One of the methods to increase production of artemisinin is by semi-synthetical production in microorganisme such as Saccharomyces cerevisiae. Semi-synthetic artemisinin in S. cerevisiae is carried out by genetically engineering of enzymes that involved in artemisinin biosynthetic pathway. Amorpha-4,11-dien synthase (ADS) is one of key enzymes in artemisinin biosynthetic pathway that catalyze reaction of farnesyl pyrophosphat (FPP) to amorphadien, an intermediete in artemisinin production. In this research, two recombinant plasmids which are (1) for expression of ADS (pBEVY-GU_ads); (2) for expression of ADS and FPS (pBEVY_GL_fps_ads) were constructed. Recombinant plasmid pBEVY- GL_fps_ads and pBEVY-GU_ads were constructed by homologous recombination method. First, ADS gen was amplified using PCR with a couple of primers which are designed in order to provide the homolog recombination of ADS gene with expression plasmid of pBEVY-GL_fps and pBEVY-GU. Template used is pUC57_ads containing synthetically optimized ADS gene. Transformants were grown in auxothropic selective media Synthetic Defined (SD) without leusin for transformants contain plasmid pBEVY-GL_fps_ads and media without uracil for transformants contain plasmid pBEVY- GU_ads. Confirmation of colonies contain plasmid recombinant was done by PCR colony with primers for amplify ADS. DNA from yeast was isolated from positive colony then transformed to E. coli. Plasmid from E. coli was isolated for restriction analysis and sequencing. Based on the restriction analysis and sequencing, it is concluded that ADS gene was successfully inserted to both pBEVY-GL_fps and pBEVY- GU plasmids.