An efficient dsRNA constitutive expression system in Escherichia coli

Area of Science:

• Molecular Biology
• Plant Science
• Biotechnology

Background:

• Synthetic double-stranded RNA (dsRNA) is crucial for reverse genetics and virus silencing.
• In vivo dsRNA synthesis in Escherichia coli (E. coli) is cost-effective and scalable compared to in vitro methods.
• Existing in vivo methods often rely on inducible T7 RNA polymerase, requiring specific inducers like IPTG.

Purpose of the Study:

• To develop a reliable vector system for efficient dsRNA synthesis in E. coli.
• To explore the use of a constitutive promoter (proD) for inducer-free dsRNA production.
• To evaluate the combined effect of constitutive and T7 promoters on dsRNA yield and gene silencing efficacy.

Main Methods:

• Engineered a vector system in E. coli featuring an insulated constitutive promoter (proD) and a T7 promoter.
• Investigated dsRNA synthesis using the constitutive promoter alone, the T7 promoter alone, and both promoters in tandem.
• Extracted bacterial RNA containing dsRNA homologous to the m5GFPer gene.
• Applied extracted dsRNA to Nicotiana benthamiana 16c plants to assess gene silencing of GFP expression.

Main Results:

• The novel vector system achieved elevated dsRNA yields without requiring inducers.
• Tandem operation of the T7 and proD promoters resulted in the highest dsRNA yield.
• Simultaneous promoter activity led to maximum metabolic cost in bacteria due to dsRNA accumulation.
• Bacterial RNA containing dsRNA from both synthetic and constitutive promoters successfully induced GFP gene silencing in plants.

Conclusions:

• The developed vector system provides an efficient and inducer-free method for high-yield dsRNA synthesis in E. coli.
• Combining constitutive and T7 promoters maximizes dsRNA production.
• Synthesized dsRNA effectively mediates gene silencing in plants, demonstrating its utility in reverse genetics and crop improvement.