#TITLE_ALTERNATIVE#

• Main Author: SEMMY NIM : 10714006 , DANIEL
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/26366
• Institution: Institut Teknologi Bandung

Ursodeoxycholic acid or UDCA is one of natural bile acid from cholesterol metabolism, which is <br /> <br /> widely used to reduce symptoms of chronic cholestatic liver diseases. The compound is commonly <br /> <br /> obtained through extraction from farm animals. Furthermore, the amount of the compound, which <br /> <br /> naturally occurs in biles is only 3% form the total bile acid. In order to generate an alternative <br /> <br /> production of UDCA, a genetic modification strategy could be applied to produce the compound in <br /> <br /> plants. This method could be done by inserting the coding gene for UDCA's biosynthesis enzymes <br /> <br /> from animals to plants through Agrobacterium sp. mediation to induce genetic modification. The <br /> <br /> cholesterol 7-&#945;-hydroxylase enzyme is the enzyme for the first biosynthesis path of UDCA from <br /> <br /> cholesterol, whose production is encoded by cytochrome P450 gene family 7 subfamily A number <br /> <br /> 1 (CYP7A1). In this research, cDNA was synthesized through reverse transcription process of <br /> <br /> ribonucleic acid (RNA) from red junglefowl (Gallus gallus) liver extraction. The cDNA was used as a <br /> <br /> template for amplification using cyp7a1 primers. The gene was ligated to pGEM®-T Easy cloning <br /> <br /> vector, which was then transformed to Escherichia coli cultured in the Luria-Bertani medium. <br /> <br /> Colonies from the transformation were selected by antibiotic screening using ampicillin and blue <br /> <br /> and white screening using IPTG and X-gal. The resulted positive colonies were cultured in liquid <br /> <br /> Luria-Bertani medium for 18 hours. Plasmids from the liquid culture were isolated and then <br /> <br /> analyzed using nucleotide base pair sequence analysis to confirm the successfulness of cyp7a1 <br /> <br /> cloning process. The nucleotide sequence was identified using the Basic Local Alignment Search <br /> <br /> Tool (BLAST®). CYP7A1 gene was successfully cloned in E. coli proven with BLAST® that shows 99% <br /> <br /> identity match with cyp7a1 mRNA accession number AY700578.1 and 99% match with cholesterol <br /> <br /> 7-&#945;-hydroxylase protein accession number AAW33560.1 <br />