STRUCTURAL DESIGN ANALYSIS OF FLOATING BRIDGES USING PONTOONS BASED ON STRENGTH AND STRUCTURAL STABILITY PLANNING ASPECTS

• Main Author: Istighozah, Azizah
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/84543
• Institution: Institut Teknologi Bandung

Reverse transcriptase (RT) enzyme plays a crucial role in converting RNA into complementary DNA (cDNA). It is widely used in molecular diagnostics and RNA-based experimental workflows. However, available commercial enzymes suffer from low processivity and thermolability. In the present study, a recombinant Moloney Murine Leukemia Virus RT (MMLV-RT) fused with Sis7a protein from Sulfolobus islandicus (MMLV-RT-Sis7a) was designed to enhance the processivity and thermostability of the enzyme. The optimal conditions for this recombinant enzyme's reaction buffer, storage buffer, and storage temperature have yet to be characterized. The reaction buffer commonly used for this enzyme is Tris-Cl-based buffer combined with other components such as KCl, MgCl2, and DTT for the reaction buffer and NaCl, EDTA, Triton X-100, and DTT for the storage buffer. An important component in the reaction buffer is KCl, which plays a role in the RNA hydrolysis reaction by RNAse H, and NaCl, which contribute to enzyme stability. The concentration of these monovalent ions need to be optimized for each RT enzyme. Therefore, in this study, buffer optimization was carried out for both ion components. Colony PCR was performed to confirm the recombinant MMLV RT-Sis7a plasmid in E. coli BL21 (DE3). Enzyme production was carried out at 25°C with 0.5 mM IPTG induction for 16 hours and visualized on SDS-PAGE gel. The soluble protein fraction was purified using Ni-NTA column and concentrated using a concentrator. Enzyme activity was tested using EnzChek™ Reverse Transcriptase Assay. First, reaction buffer determination was carried out with variations in KCl concentration (50, 75, and 100 mM) combined with all variations of the storage buffer with variations in NaCl concentration (50, 100, and 150 mM). The optimal reaction buffer was then used for storage buffer testing for 3 months (sampling points 0, 2, 4, 8, and 12 weeks) stored at -20, 4, and 25°C. The recombinant plasmid was successfully confirmed with an amplicon size of ~2.5 kb. The enzyme was successfully produced, purified, and concentrated, as indicated by the presence of a protein band of ~82 kDa on the SDS-PAGE gel. Based on the fluorescence data, the optimal reaction buffer was 50 mM KCl. However, all three variations of NaCl concentration used as storage buffer caused a decrease in fluorescence during testing for 3 months, so the optimal NaCl concentration was chosen based on the lowest percentage decrease in fluorescence, namely 50 mM NaCl stored at -20°C with an average decrease of 22.61%. Therefore, this study obtained the optimal reaction buffer for the MMLV-RT-Sis7a enzyme, but further research is needed on the storage buffer components. In addition, variations in other buffer components that can be varied and have not been done in this study need to be investigated.