In situ massive production of double-stranded RNAs from escherichia coli

Abstract

Since its first discovery of RNA interference (RNAi), the technology has been widely applied for the genetic engineering of various organisms. Application nowadays of RNAi requires extensive amount of double-stranded RNA (dsRNA) as the inducer of gene silencing, thus the method for massive production of dsRNA should be established. In this thesis, a novel in situ synthesis of dsRNA was suggested for the increased titer. For this, T7 RNA polymerase was employed for the synthesis of final product from the production host, Escherichia coli BL21(DE3), and lytic proteins originated from bacteriophages was introduced for the leakage of this protein. RNAse inhibitor was also introduced for the stability of end product, and every engineering was done in host genome. By fed-batch fermentation of this strain, the titer of dsRNA from culture sample was obtained as 207 mg/L culture, which shows higher production rate than previous reports.