An efficient dsRNA constitutive expression system in Escherichia coli

Abstract

Synthetic dsRNA are valuable tools for reverse genetics research and virus silencing applications. Its synthesis can be performed both in vivo or in vitro. Whilst the latter presents the drawback of high production cost, the former has the advantage of being less expensive and suitable for scalable production. In general, dsRNAs are obtained in vivo from Escherichia coli heterologous systems that require the gene for the T7 RNA polymerase inducible by IPTG. The (ds)RNAs for gene of interest are then synthesized under the T7 promoter. In this work, we present a reliable vector system that includes the insulated promoter proD for the constitutive expression of dsRNA in E. coli that does not require any inducer and that renders elevated dsRNA yield. In tandem, the T7 and proD promoters render the highest dsRNA yield. The accumulation of dsRNA in this system entails a high metabolic cost for the cell. Bacterial RNA extractions that included dsRNAs homologous to the m5GFPer gene and derived from both the synthetic and constitutive promoters induce silencing of GFP expression in Nicotiana benthamiana 16c.Key points• A vector system that includes a constitutive promoter and a T7 promoter in tandem for maximizing dsRNA synthesis.• The metabolic cost for bacteria is maximum when the two promoters are operating simultaneously and results from the accumulation of dsRNA.• Bacterial RNA extractions from both the induced and constitutive systems that include a mGFP5er-derived dsRNA are capable of silencing the GFP expression in Nicotiana benthamiana 16c plants.

Keywords: Constitutive promoter; D-lactose; Escherichia coli; GFP silencing; IPTG; In vivo synthesis; dsRNA synthesis.