Part:BBa_K5186017
pTDH3-shRNA(shaker)-tTDH1
Description
pTDH3-shRNA(shaker)-tTDH1 is an expression cassette in E. coli expressing shRNA(shaker) (BBa_K5186015), which is a short hairpin RNA targeting mosquitoes' Shaker gene. With this expression cassette, shRNA(shaker) can be constitutively expressed under the control of pTDH3(BBa_K124002) and tTDH1 (BBa_K2753052) in S. cerevisiae. It can be used for the preparation of RNAi-based mosquitocides.
This is a part of a part collection containing shRNAs targeting mosquitoes' vital survival genes including 5-HTR1, Rbfox1, Shaker and Irx (BBa_K5186011, BBa_K5186012, BBa_K5186013, BBa_K5186014, BBa_K5186015, BBa_K5186016, BBa_K5186017). This collection serves as a valuable resource for the iGEM community and researchers, offering a safe, efficient, and environmentally friendly approach to mosquito control.
Usage and Biology
The gene Shaker (AAEL000242) encodes an evolutionarily conserved subunit of voltage-gated potassium channels, which function as critical regulators of neural and muscular development in divergent species. Interference of this gene leads to impaired nerve function, resulting in paralysis and deaths of the mosquito. (Keshava M. et al, 2020)
Recognizing the significance of the Shaker gene to mosquitoes, shRNA(shaker) (BBa_K5186015) was designed to target the Shaker gene, referring to Sh.463 target site (Corey B. et al, 2023). Upon expression of this cassette in S. cerevisiae CEN. PK2-1C, the yeast cells can be transformed into RNAi-based mosquitocides after inactivation and freeze-drying.
Once digested by mosquitoes, the shRNA(shaker) will be processed into small interfering RNAs (siRNAs), and further specifically target mRNA(Shaker) to achieve degradation. This targeted interference results in neural misfunctions in mosquitoes, eventually causing their deaths without any harm to other non-target organisms.
Figure 1. Developing process of RNAi yeast mosquitocides.
Characterization
In the end, 6 shRNAs were engineered to target mosquitoes' vital survival genes (Figure 2a, 2b). To validate the successful production of our shRNAs in yeast, we extracted RNA from S. cerevisiae and conducted electrophoresis. And the designed shRNAs are 55 nts with poly T ending sequence, which corresponds to the electrophoresis result, indicating the successful generation of shRNAs (Figure 2c).
In our experimental validation, mosquitoes were divided into a control group and an experimental group. The experimental group was provided with freeze-dried, inactivated yeast cells engineered to express a variety of shRNAs, including shRNA1 (5-HTR1), shRNA2 (5-HTR1), shRNA3 (5-HTR1), shRNA (Rbfox1), shRNA (Shaker), and shRNA (Irx). Over a five-day observation period, we monitored the impact of these shRNAs on mosquito survival. (Figure 2b)
Our findings revealed that while the control group exhibited a natural mortality rate of 5%, all experimental groups experienced a complete 100% mortality rate by the 3rd day after feeding. This outcome not only confirms the potency of our RNAi-based mosquitocides but also underscores their rapid effect, holding significant promise for the swift control of mosquito populations.
Figure 2. 6 variants of shRNAs targeting mosquitoes' vital survival genes are expressed in S. cerevisiae CEN. PK2-1C. (a) Mosquitoes' vital survival genes 5-HTR1, Rbfox1, Shaker, Irx are chosen to silence, encoding for serotonin receptor, RNA binding proteins, voltage-gated potassium channels and Iroquois-class homeodomain-containing proteins respectively. They involve critical functions including neural, immune, reproductive and muscular development. (b) Genetic circuit and nucleotide sequences of shRNAs expression. (c) Gel electrophoresis analysis of RNA extracted from yeast cells expressing various shRNAs. (d) Mortality of mosquitoes consuming freeze dried inactivated yeast cells expressing various shRNAs.
Reference
Mysore, K., Njoroge, T.M., Stewart, A.T.M. et al. (2023). Characterization of a novel RNAi yeast insecticide that silences mosquito 5-HT1 receptor genes. Sci Rep 13, 22511 . https://doi.org/10.1038/s41598-023-49799-3
Mysore, K., Longhua, S., Limb, K.H. et al. (2022). A broad-based mosquito yeast interfering RNA pesticide targeting Rbfox1 represses Notch signaling and kills both larvae and adult mosquitoes. Pathogens, 11(9), 956. https://doi.org/10.3390/pathogens11090956
Corey B., Keshava M., Teresia M. N., Seth McConnell, et al. Targeting Mosquitoes through Generation of an Insecticidal RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae. J. Fungi. 2023, 9, 1056. https://doi.org/10.3390/jof9111056
Keshava M., Longhua S., Limb K. H., et al. A Yeast RNA-Interference Pesticide Targeting the Irx Gene Functions as a Broad-Based Mosquito Larvicide and Adulticide. Insects. 2021, 12: 986. https://doi.org/10.3390/insects12110986
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 738
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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