Optimizing dsRNA engineering strategies and production in E. coli HT115 (DE3)

Author:

da Rosa Juliana12ORCID,Viana Américo José Carvalho23,Ferreira Fernando Rafael Alves23,Koltun Alessandra2,Mertz-Henning Liliane Marcia2,Marin Silvana Regina Rockenbach2,Rech Elibio Leopoldo4,Nepomuceno Alexandre Lima2

Affiliation:

1. Department of General Biology, Londrina State University , Celso Garcia Cid Road, PR 445, km 380, University Campus, 86057-970 Londrina, PR , Brazil

2. Embrapa Soja, Carlos João Strass Highway, Acess Orlando Amaral, District of Warta , 86085-981 Londrina, PR , Brazil

3. Arthur Bernardes Foundation , Headquarters Building, no number - University Campus, 36570-900 Viçosa, MG , Brazil

4. Embrapa Genetic Resources and Biotechnology, National Institute of Science and Technology in Synthetic Biology , 70770-917 Brasilia, DF , Brazil

Abstract

Abstract   Producing double-stranded RNA (dsRNA) represents a bottleneck for the adoption of RNA interference technology in agriculture, and the main hurdles are related to increases in dsRNA yield, production efficiency, and purity. Therefore, this study aimed to optimize dsRNA production in E. coli HT115 (DE3) using an in vivo system. To this end, we designed a new vector, pCloneVR_2, which resulted in the efficient production of dsRNA in E. coli HT115 (DE3). We performed optimizations in the culture medium and expression inducer in the fermentation of E. coli HT115 (DE3) for the production of dsRNA. Notably, the variable that had the greatest effect on dsRNA yield was cultivation in TB medium, which resulted in a 118% increase in yield. Furthermore, lactose induction (6 g/L) yielded 10 times more than IPTG. Additionally, our optimized up-scaled protocol of the TRIzol™ extraction method was efficient for obtaining high-quality and pure dsRNA. Finally, our optimized protocol achieved an average yield of 53.3 µg/mL after the production and purification of different dsRNAs, reducing production costs by 72%. One-Sentence Summary Improved production of double-stranded RNA (dsRNA) in E. coli HT115 (DE3) bacteria resulted in significant yield increases and cost reductions through optimized methods.

Funder

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Publisher

Oxford University Press (OUP)

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