Contribution

Overview

Double-stranded RNA (dsRNA) production has been a prominent feature in numerous past iGEM projects, showcasing its potential across various applications. Notably, the Lanzhou_2017 and Ecuador_2021 iGEM teams harnessed bacteria as a chassis to generate dsRNA, effectively employing it as biopesticides within the agricultural sector. Our project aims to synthesize a dsRNA specifically designed to combat Rhizoctonia solani, a fungus responsible for corn sheath blight. By developing a targeted approach, we aim to prevent and treat this disease, thereby enhancing both the yield and quality of corn crops. DsRNA is increasingly recognized for its environmental sustainability and as a promising alternative to conventional chemical pesticides (Hough et al., 2022). However, the efficacy of both pesticides and fungicides in this context hinges on the production of dsRNA at a scale that is economically viable (Dalakouras et al., 2024). A critical challenge that must be addressed is the need for cost-effective dsRNA production. The production process is broadly divided into two phases: prokaryotic expression and subsequent extraction. Controlling costs during the prokaryotic expression phase often involves strategic selection of vectors and expression strains, as detailed in the literature(Ma et al., 2020; Yin et al., 2009). In this project, we have focused on optimizing the dsRNA extraction process, striving to establish a method that is not only cost-effective but also highly efficient. We aspire for our contributions to serve as a valuable resource for future iGEM teams endeavoring to produce dsRNA.

Comparison of extraction methods for prokaryotic expressed dsRNA

Several methods have been described for dsRNA extraction from bacterial cells, like TRIzol method, phenol chloroform method, RNA-easy extraction method and alcohol precipitation method (Rui et al., 2021). We compare these methods in the table below.

Table 1 Comparison of four dsRNA extraction methods

Among these four methods, the phenol-chloroform-based protocol has been reported to be an effective method for extracting dsRNA with high yield and quality. However, for the health of our team members and to comply with the safety policies in the iGEM Competition, we have excluded the TRIzol method and the phenol chloroform method. As a result, we would have to decide between RNA-easy extraction method and ethanol precipitation method.

Our team’s efforts

We used L4440-HT115 (DE3), a widely used dsRNA production system, to express our target dsRNA. First, construct the RsCAT-dsRNA-L4440 recombinant expression vector (Fig. 1A), then transform the expression strain HT115 (DE3) (Fig. 1B) and screen the positive clones by colony PCR (Fig. 1C).

Fig. 1 Obtaining HT115 (DE3) positive transformation strain. (A) The recombinant plasmid RsCAT-dsRNA-L4440 map. (B) The recombinant plasmid was transformed into E. coli HT115(DE3) competent cells and coated with bacterial culture medium for overnight culture. (C) Verify positive colonies by colony PCR.

In order to increase the yield of dsRNA, we optimized the induction concentration of IPTG. First, we set up different concentration gradients of IPTG for induction. Then, agarose gel electrophoresis was performed and dsRNA concentration was determined using a micro-spectrophotometer (Fig. 2). The results showed that the yield of RsCAT-dsRNA was the highest when the IPTG induction concentration was 0.5 mM.

Fig. 2. Yield identification of dsRNA induced by different concentrations of IPTG. The dsRNA bands are approximately 310 bp in size and their concentrations are indicated below the bands. M: Marker；1: 0 mM IPTG；2: 0.05 mM IPTG；3: 0.2 mM IPTG；4: 0.5 mM IPTG；5: 1 mM IPTG.

Next, we cultured 50 mL of bacterial culture until the OD was between 0.6 and 0.8, added IPTG with a final concentration of 0.5 mM to induce expression at 37°C for 3 h, and collected the bacterial precipitate. We extracted dsRNA using alcohol precipitation method and RNA-easy extraction method to extract dsRNA respectively. For detailed extraction protocols, please refer to [Experiments].

The results of agarose gel electrophoresis showed that the dsRNA extracted by both methods had obvious bands at 310 bp. The band of dsRNA extracted using the alcohol precipitation method was more dispersed than the one extracted using the RNA-easy extraction method, but there were no obvious non-target bands overall (Fig. 3A&3B). By using a micro-spectrophotometer to detect and compare the test results (e.g. concentration, OD260/280), we found that there was not much difference in the yield and purity of the dsRNA extracted by the two methods (Fig. 3C&3D).

Fig. 3 The dsRNA extracted by the two methods was verified by agarose gel electrophoresis and microspectrophotometer. (A, C) dsRNA extract by alcohol precipitation method. (B, D) dsRNA extract by RNA-easy extraction method.

Conclusion

The dsRNA we extracted will be used in the agricultural field. As a fungicide, it needs to be sprayed on corn plants in large quantities to prevent and control sheath blight. Therefore, cost is our priority. In view of the simple extraction steps and low reagent cost of the alcohol precipitation method, it is considered as the preferred method for extracting dsRNA for prokaryotic expression.

References

[1] Dalakouras, A., Koidou, V., & Papadopoulou, K. (2024). DsRNA-based pesticides: Considerations for efficiency and risk assessment. Chemosphere, 141530.

[2] Hough, J., Howard, J. D., Brown, S., et al. (2022). Strategies for the production of dsRNA biocontrols as alternatives to chemical pesticides. Frontiers in Bioengineering and Biotechnology, 10, 980592.

[3] Ma, Z.-Z., Zhou, H., Wei, Y.-L., et al. (2020). A novel plasmid–Escherichia coli system produces large batch dsRNAs for insect gene silencing. Pest Management Science, 76(7), 2505-2512.

[4] Rui, C., Jun, W., Kaiyun, F., et al. (2021). Comparative Study on the Effect of 4 kind of dsRNA Extraction Methods form Prokaryotic Expression Double-stranded RNA. Xinjiang Agricultural Sciences, 58(4), 700-711.

[5] Yin, G., Sun, Z., Liu, N., et al. (2009). Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system. Applied Microbiology and Biotechnology, 84(2), 323-333.