ISOLATION AND CLONING OF ENZYME ALDH1 GENES FROM ARTEMISIA ANNUA L. PLANT

• Main Author: Luthfiana Rizwan, Yayang
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45475
• Institution: Institut Teknologi Bandung

Malaria is a serious endemic disease that threatens more than one third of the global population and kills approximately 1.238.000 people annually. During the latest decade, Artemisia annua (L.) has received increasing attention due to the fact that the plant produces the sesquiterpenes lactone endoperoxide artemisinin, which today is widely used for the treatment of malaria. Nowadays the major method to get artemisinin is only through isolation from it’s plant source, however, artemisinin production naturally in it’s plant source is relatively low (0,01–0,5%). It makes artemisinin, a drugs which is used for therapy of malaria disease, has higher demand than its supplies. Many research has been done in order to reveal the biosynthetic pathway of artemisinin and the enzymes that play a role on it. Due to the lack of artemisinin that could be isolated from it’s plant source, it is necessary to combine bioengineering technique in order to construct a recombinant plasmid that contain an inserted genes which is encode the enzymes that play a role on artemisinin biosynthetic pathway, aldehyde dehydrogenase 1 (ALDH1), with tissue culture technique to increase the artemisinin production. Recombinant plasmids construction was started through RNA isolation from the A. annua plant by using TRIzol method which include 5 phase, homogenization phase, separation phase, precipitation phase, RNA washing and dilution and then confirmation by gel electrophoresis and visualization using UV transilluminator. Then, cDNA was sythesized by reverse transcription mechanism used reverse transcriptase enzyme, which is confirmed by using Polimerase Chain Reaction (PCR) methods that used actin specific primer to produce 200 bp size PCR product. PCR was performed to the cDNA through touchdown PCR by using ALDH1 specific primer, which has been constructed based on enzyme ALDH1 genes (annealing temperature 50 –60 o C) to produce 1500 bp size PCR product. Then the PCR product was purified by using Geneaid® Gel/PCR DNA Fragments Extraction Kit which include disintegration gel, DNA binding, DNA washing and elution and then confirmation by using gel electrophoresis. Then to the DNA Fragment was performed ligation into the pJET 1.2/blunt vector by using CloneJET™PCR Cloning Kit. The recombinant plasmid was transformed into competent E. coli DH5?through heat shock methods with incubation in 42 o C. After that, the transformation product (transforman) was grown on liquid LB media which was incubated in 37 o C by using shaker incubator first. Then the transforman was grown in solid ampicilin resistant selective LB media with incubation in 37 o C for 16-18 hours long. Then the recombinant plasmid was isolated from the E. coli DH5?culture cells by using Geneaid® High-Speed Plasmid Mini Kit which include transforman harvesting, resuspension, lysis, neutralization, DNA binding and elution. The presence of inserted genes, was confirmed by performing PCR that used ALDH1 specific primer. restriction analysis using NdeI restriction enzyme, was also performed in order to confirm the presence of inserted genes. The result of performing PCR and restriction analysis showed that the gene ALDH1 has been inserted into plasmid. However, the result of DNA sequencing has not succeed yet in confirming the inserted gene in plasmid pJET 1.2/blunt.