OPTIMIZATION OF HIGH DENSITY CULTURE ESCHERICHIA COLI TOP10 TO INCREASE PLASMID DNA PRODUCTION AS A TEMPLATE FOR RNA SYNTHESIS IN THE IN VITRO TRANSCRIPTION PROCESS

• Main Author: Aulia Afifah, Salma
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/74205
• Institution: Institut Teknologi Bandung

The development of mRNA is increasingly interesting to study, especially in ex vivo and in vivo therapy. mRNA is widely used for the development of gene editing-based therapies and mRNA vaccines. The aim of this study was to optimize the medium to achive high density culture and harvest time to increase plasmid DNA production as in vitro transcription template for mRNA production. This study used a medium modification approach to achieve high density culture Escherichia coli TOP10 pGEM-T_N1_nCoV in batch cultivation method. The medium were LB; LB with phosphate buffer (K2HPO4 12.549 g/L and KH2PO4 2.31 g/L); LB with glycerol (50 g/L); LB with glycerol and phosphate buffer; LB with phosphate buffer, glycerol, glucose (15 g/L), and galactose (15 g/L). The effect of additional carbon sources and phosphate buffer was observed by measuring OD600 and wet cell weight. The highest OD600 and wet cell weight was obtained from culture in LB with glycerol and phosphate buffer, with OD600 of 4.78 ± 0.14 and wet cell weight 36 ± 0.63 mg/mL. Plasmid DNA was isolated from the culture after 5 and 7.5 hours incubation. Calculation of plasmid DNA concentration was carried out by measuring OD260 and semi-quantitative analysis from agarose gel electrophoresis. The LB medium with glycerol and phosphate buffer increased the volumetric concentration of plasmid DNA of 1516.97 ± 385 ng/mL after 5 hours incubation. The in vitro transcription performed using this plasmid resulted in 0.9228 ng RNA/ng DNA. High density culture E. coli TOP10 pGEM-T_N1_nCoV was achieved by addition of glycerol and phosphate buffer to LB medium and increasing yield volumetric of plasmid DNA after 5 hours incubation.