RECOMBINANT DSRNAI PLASMID PRODUCTION IN ESCHERICHIA COLI GT115 IN BAFFLED-FLASK AND BIOREACTOR

• Main Author: Wijayana, Sena
• Format: Theses
• Language: Indonesia
• Subjects: Teknologi
• Online Access: https://digilib.itb.ac.id/gdl/view/72690
• Institution: Institut Teknologi Bandung

Application of plasmid coding synthetic dsRNA targeted IMNV genome (Infectious Myonecrosis Virus) can reduce viral replication in shrimp industry with activating RNA interference (RNAi) response. Application of dsRNAi plasmid as antiviral for IMNV in shrimp-farm need a huge quantity of plasmid. Bioreactor can be used for large-scale plasmid production to achieve high plasmid yield. Plasmid production in bioreactor can be improved by selection of the fermentation media and the fermentation strategy. This research aim is to determine the fermentation media and fermentation strategy to produce recombinant dsRNAi plasmid with high plasmid yield and cost-effective plasmid production. Plasmid production was conducted in Escherichia coli GT115 bearing recombinant dsRNAi plasmid in Luria Bertani media with addition of buffer solution (LB+), Terrific-Broth (TB) and M9+Modification (M9+Mod). The fermentation process in media selection stage was conducted at 37oC, 150 rpm agitation, 150 mL culture volume, with ampicillin addition in 250mL baffled-flask. Fermentation media with the highest plasmid yield and cost-effective in production media cost was used for large-scale production in 10L bioreactor. Production in bioreactor was conducted with batch and fed-batch fermentation strategy (feeding media contain 400 g/L glycerol, 80 g/L tryptone, and 40 mL MgSO4 1M). Fermentation process in bioreactor was set at 30oC, pH 7.0, 7 L culture volume and 100-300 rpm agitation. Temperature was set at 30oC to reduce metabolic burden during fermentation process. Then, plasmid was confirmed by gel agarose electrophoresis, PCR (Polymerase Chain Reaction) and sequencing the insert gene. As a result, plasmid production in TB media has higher biomass growth rate and plasmid production rate than production in M9+Mod and LB+ media. Plasmid production in TB media in baffled-flask resulted plasmid yield in 2.318 mg/L, 14-fold higher than M9+Mod (0.165 mg/L), and 34-fold higher than LB (0.068 mg/L) with plasmid production cost was Rp.79.34/?gplasmid, 7-fold lower than M9+Mod (Rp587.7/?gplasmid) and 15-fold lower than LB+ (Rp1453.80/?gplasmid). In bioreactor production, addition of feeding media can increase the time period of biomass growth and plasmid production still in logarithmic phase. Plasmid production in fed-batch fermentation in bioreactor resulted plasmid yield in 10.186 mg/L, 5-fold higher than batch fermentation (1.882 mg/L) with plasmid production cost was Rp28.59/?gplasmid, 3-fold lower than batch fermentation (Rp93.87/?gplasmid). Plasmid was confirmed in agarose gel electrophoresis in ~5750 bp DNA band after restriction, and insert gene was confirmed in 700 bp DNA band. In conclusion, dsRNAi plasmid production in Escherichia coli GT115 with high-yield and cost-effective plasmid production can be achived in TB media with fed-batch fermentation strategy. Fermentation was carried out at 30oC, pH 7.0 and 100-300 rpm agitation.