Media Selection and Fed-Batch Fermentation Strategy for dsRNA Plasmid Production in Escherichia coli GT115

Abstract

Application of plasmid encoding synthetic dsRNA targeted IMNV genome (Infectious Myonecrosis Virus) can reduce viral replication in the shrimp industry by activating RNA interference (RNAi) response. Application of dsRNA 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 the bioreactor can be improved by selection of the host organism, the recombinant plasmid vector, the fermentation media, and the fermentation strategy. This research aim is to determine the fermentation media and fermentation strategy to produce recombinant dsRNA plasmid with high plasmid yield. Selection of fermentation media was conducted in a baffled flask with three different media. Then, the optimum media was used for optimization in bioreactor production with the addition of feeding media. As a result, plasmid production in TB media has a 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 in 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). In bioreactor production, plasmid production in fed-batch fermentation in bioreactor resulted plasmid yield in 1.018 mg/L, 5-fold higher than batch fermentation (1.882 mg/L). Plasmid was confirmed in agarose gel electrophoresis at [~]5750 bp and insert gene at 700 bp. The cultivation technique developed should be workable for the pilot scale. Downstream processing in plasmid production should be able to achieve plasmid with high concentration and purity.