KONSTRUKSI PLASMID pETDUET_CYP_ADS_FPS UNTUK SISTEM EKSPRESI DI ESCHERICHIA COLI

• Main Author: Novita Balqis, Rana
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/44576
• Institution: Institut Teknologi Bandung

Artemisinin is a sesquiterpene lactone produced by Artemisia annua L. which is potential as an anti-malarial drug and anti-hepatitis B. Artemisinin is synthesized in low quantities by its original plant. Hence, other approach are needed to obtain artemisinin in large amount. One of the potential method is addition of artemisinic acid (precursor of artemisinin) biosynthesis pathway by genetic engineering on the enzymes involved in the biosynthesis of artemisinic acid, such as farnesyl pyrophosphate synthase (FPS), amorpha-4,11-diene synthase (ADS), and cytochrome P450 monooxygenase (CYP) in Escherichia coli. The purpose of this research is to construct pETDUET_CYP_ADS_FPS plasmid by cloning FPS gene into pETDUET vector which already had ADS dan CYP. The FPS gene was obtained from pJexpress401_FPS by using PCR method and produced FPS gene which is including restriction site of Not?ð?­??í ?????¸ ??????±?¸??íð?»? ã?±??? ? cloned by ligation method to pETDUET_CYP_ADS vector that had been cut by the same enzyme, NotI. Confirmation of the recombinant vector was done by migration, restriction site, PCR product, and sequencing analysis. Confirmed pETDUET_CYP_ADS_FPS was transformed into Escherichia coli BL21(DE3) for confirmation of each protein production ability. í± ??¸?®?ð???? results were analyzed by SDS-PAGE showed a ????±ð???­ ?¸? ??í± ?ð?± ??????839 kDa (FPS), 53.42 kDa (CYP), dan 59.98 kDa (ADS). Those proteins were detected when induced with IPTG at final concentration 0.1 mM and 0.5mM with 4 and 5 hours induction time, and final concentration 1 mM with 5 hours induction time. Optimal condition for those proteins was when induced with IPTG at final concentration 1 mM with 5 hours induction time.