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

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%.

Keywords: Bacterial dsRNA production; Double-stranded RNA; Lactose induction; RNAi.

Graphical Abstract

Fig. 1.

Expression vectors and dsRNA production in E. coli HT115 (DE3). (a) Scheme of the L4440 vector showing the multiple cloning site and bidirectional T7 promoters. (b) Scheme of pCloneVR_2 showing the multiple cloning site flanked by bidirectional T7 promoters and terminators. (c) Schematic diagram of dsRNA production in the E. coli HT115 (DE3) system. The target gene fragment is inserted into the multiple cloning site of L4440 or pClone_VR_2. Next, the vector is transformed into E. coli HT115 (DE3), an RNase III-deficient strain. Bacteria grow in a liquid medium, and with the addition of the inducer, the expression of T7 RNA polymerase occurs. Next, T7 polymerase transcribes the dsRNA strands. Subsequently, the produced dsRNA can be extracted and purified. *MCS = multiple cloning site.

Fig. 2.

Production of dsRNA from the PDS gene using the vectors (a) L4440 and (b) pCloneVR_2 produced with E. coli HT115 (DE3) system. M: 1 kb plus DNA ladder.

Fig. 3.

dsRNA extraction using TRIzol^TM and ethanol extraction methods. (a) Agarose gel after dsRNA extraction from liquid fermentation in the HT115-L4440 system and lactose induction. M: 1 kb plus ladder; Lanes 1, 2, and 3 are the triplicates of ethanol extraction; Lanes 4, 5, and 6 are the triplicates of TRIzol^TM extraction; Lanes 7, 8, and 9 are the triplicates after treatment with DNase and RNase A; Lanes 10, 11, and 12 are the triplicates after DNase and RNase A treatment. The arrow indicates a dsRNA size of approximately 341 bp. (b) Yield of dsRNA-PDS after extraction with TRIzol^TM and ethanol extraction methods. * indicates a significant difference according to Student's t-test, p < .05.

Fig. 4.

Expression inducers IPTG and lactose for dsRNA production in E. coli HT115 (DE3) using the vector pCloneVR_2. (a) dsRNA yield comparison different lactose concentrations and IPTG as inducers, analyzed using the Tukey test. (b) dsRNA yield comparison between induction at the beginning of fermentation and after reaching an OD₆₀₀ of 0.4, analyzed using the Student's t-test. Error bars represent the standard deviation of the mean. * indicates a significant difference (p < .05).

Fig. 5.

Comparative analysis of dsRNA yield between LB, TB, and M9 media in the HT115/pCloneVR_2 system using the inductor concentration of 6 g/L and upscale TRIzol ^TM method. (a) Yield of dsRNA-EPSPS in the three media tested. (b) dsRNA quality after extraction and purification. (c) OD600 values during fermentation. M: 1 kb plus ladder. * indicates a significant difference according to Tukey's test, p < .05.

Fig. 6.

Yield and quality of different dsRNAs produced using the HT115 (DE3)/pClone_VR_2 system under optimized cultivation and extraction conditions. The black line represents the arithmetic mean of the yield of all dsRNAs (53.3 µg/mL). (b) The 260/280 and 260/230 ratios measured on a Nanodrop spectrophotometer.