ISOLASI DAERAH PENGKODE TATAD DAN TATCD DARI BACILLUS SUBTILIS ATCC 6633 MENGGUNAKAN METODE POLYMERASE CHAIN REACTION

• Main Author: Sulistiawati, Ayu
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/51507
• Institution: Institut Teknologi Bandung

Recombinant protein expressed intracellularly in Escherichia coli is often result in the formation of inclusion bodies. Inclusion bodies can be minimalized by targeting recombinant protein to periplasmic space. Recombinant protein can be targeted into periplasmic space by using Bacillus subtilis’s Twin-Arginine Translocation (TAT) AdCd. In this research, tatAd and tatCd Coding Sequence (CDS) were isolated from Bacillus subtilis ATCC 6633 using Polymerase Chain Reaction (PCR) method. tatAdCd CDS will be cloned into expression vector and is expected to help transporting recombinant protein to periplasmic space in further research. Before isolation of tatAd and tatCd are carried out, an in silico design for primer PCR is needed. Primers are designed for production of tatAd and tatCd in the polycistronic form using native Ribosom Binding Site (RBS) and synthetic RBS from the primer that has been optimized for expression in E. coli. There are four primers designed for this research. Primers have been analyzed for its specificity, self dimer, pair dimer, and hairpin formation. tatAd and tatCd CDS are isolated by PCR in two separate tubes. tatAd and tatCd CDS that have been isolated then purified by gel extraction method. Purified tatAd and tatCd CDS are put together in the same tube to generate tatAdCd CDS fragment with optimized RBS by PCR. tatAd, tatCd, and tatAdCd CDS are isolated by PCR under the following conditions: predenaturation (94?C, 5 minutes), denaturation (94?C, 30 seconds), annealing of the primer (45 seconds at 58?C for isolation tatAd and tatAdCd, at 54?C for isolation tatCd), and elongation (68?C, 45 seconds). PCR is performed in 25 cycles using KOD polymerase and ended by final elongation at 68?C for 3 minutes. The PCR products were confirmed by DNA electrophoresis using 1% agarose gel, at 80 V, 400mA, for 60 minutes. The result showed that tatAd, tatCd, and tatAdCd were successfully isolated in the presence of DNA band at a size of 251, 768, and 1034 bp, respectively.