CLONING FERREDOXIN, FLAVODOXIN AND FERREDOXIN (FLAVODOXIN) REDUCTASE GENE INTO PET16B EXPRESSION VECTOR.

• Main Author: Jessica
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/78416
• Institution: Institut Teknologi Bandung

In order to improve the production of artemisinin, efforts were made to produce artemisinin and other terpenoids semisynthetically by incorporating microbial genetic engineering as an alternative for sustainable production. The promiscuity of amorphadiene synthase allows the enzyme to utilize hydroxylated farnesyl (12-OH FPP) as a substrate to produce artemisinic aldehyde. This process led to a shortened artemisinin biosynthesis pathway. CYP124 protein from Mycobacterium tuberculosis is known to have the ability to catalyze ?-hydroxylation reaction on FPP. However, CYP124 requires protein carriers and electron transfer from NADPH/NADH, namely ferredoxin or flavodoxin and ferredoxin (flavodoxin) reductase. This research was conducted to clone the genes that code for these three proteins and insert them into the pET16b expression vector through QuickStep-Cloning and Circular Polymerase Extension Cloning (CPEC). The QuickStep-Cloning has not been successful because of the difficulty of obtaining single-strand DNA from asymmetric PCR. Gene cloning using the CPEC method was successfully transformed into Escherichia coli DH5? competent cells. The success of gene cloning was confirmed through colony PCR, restriction analysis, and DNA sequence analysis. The fpr gene has successfully been inserted into pET16b. The research phase was continued with protein expression experiments at 37oC with IPTG induction and without IPTG induction.