Overview
2024 AIS-China targets to produce HMBPP, a blood-feeding attractant, by genetically modifying E. coli and specific mosquitocide shRNAs by engineering S. Cerevisiae. Integrating HMBPP and shRNA mosquitocides with traditional sugar baits, a novel ATSB is borne for mosquito-borne diseases, which is named Moskilla hereby. We aim to provide a promising new solution, which is not only eco-friendly and safe but also effective and specific to mosquito populations, for the prevention and control of vector borne diseases. This year our team has achieved the following contributions below.
Part collection for HMBPP overproduction
In our pursuit to enhance HMBPP production, we adopted 2 strategies: overexpressing key rate-limiting enzymes of the MEP pathway and downregulating the expression of the downstream gene IspH using synthetic small RNAs (sRNAs).
For the overexpression approach, we utilized existing parts to engineer a series of expression combinations, resulting in the development of various MEP overexpression cassettes (BBa_K5186006, BBa_K5186007, BBa_K5186008, BBa_K5186009). Utilizing the lycopene synthesis cassette (BBa_K274100) as a reporter, we demonstrated BBa_K5186008 a 2.03-fold increase in overexpression efficiency, marking it as the optimal choice for the MEP overexpression cassettes within our part collection. We have added the characterization documentation for existing parts such as DXS (BBa_K3166061), DXR(BBa_K1950003), IspG (BBa_K1088004), and IspDF (BBa_K1653001).
Based on the overexpression results, the integration of sRNA2(IspH) (BBa_K1653005) leads to the highest yield of HMBPP without affecting bacterial growth in E. coli. We have built a part collection that serves as a valuable resource and inspiration for other iGEM teams and researchers, aiming to achieve higher yields of HMBPP or other isoprenoids in E. coli.
Figure 1. Using lycopene as reporter, the best MEP overexpression cassette is selected for higher yield of HMBPP. (a) Various MEP pathway overexpression cassettes expression in E. coli strain DH5a (b) Production of lycopene via the endogenous MEP pathway in E. coli. (c) Gel electrophoresis analysis of transformed MEP pathway overexpression cassettes. (d) Relative lycopene production while using various MEP Overexpression Cassettes in E. coli.
Figure 2. 4 variants of sRNA(IspH)s are engineered in E. coli for down-regulation of IspH. (a) A graphical abstract of the molecular mechanism underlying the down-regulation of IspH expression by sRNA(IspH)s. (b) Genetic circuit and nucleotide sequences of sRNA(IspH)s expression. The green and blue sequences indicate the target-binding sequences and SgrS-S scaffold variants respectively. (c) Gel electrophoresis analysis of transformed sRNA(IspH)s expression cassettes. (d) Growth curve of control (E. coli strain DH5a) and strains expressing sRNA(IspH)s.
Figure 3. Analysis of HMBPP production when expressing various sRNA(IspH)s. (a) Standard curve of HMBPP. (b) The yield of HMBPP in NC, PC, sRNA1-4 fermentation products.
Note: The fact that the HMBPP concentration in the NC product is 0 does not imply the absence of HMBPP; rather, it suggests that its concentration is below the LC-MS instrument's detection threshold.
Targeted shRNA Part Collection for Efficient Mosquito Control
To address mosquito populations, we have engineered variants of short hairpin RNAs (shRNAs) targeting essential survival genes in mosquitoes, including 5-HTR1, Rbfox1, Shaker, and Irx. By employing RNA interference (RNAi) techniques, these shRNAs effectively silence the targeted genes, leading to the death of the mosquitoes without any harm to other non-target organisms. These shRNAs (BBa_K5186011, BBa_K5186012, BBa_K51860013, BBa_K5186014, BBa_K5186015, BBa_K5186016, BBa_K5186017) contribute to mosquitoes control solutions and thus make up a part collection.
Figure 4. 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, e) Survival curve of mosquitoes consuming freeze dried inactivated yeast cells expressing various shRNAs.
Note: 1-6 indicates expression cassettes of shRNA1 (5-HTR1), shRNA2 (5-HTR1), shRNA3 (5-HTR1), shRNA (Rbfox1), shRNA (Shaker), respectively.
We have successfully validated that adult mosquitoes exposed to this shRNA part collection achieve a 100% mortality rate by the third day, highlighting the potential of shRNAs as RNAi-based mosquitocides. This collection serves as a valuable resource for the iGEM community and researchers, providing a safe, efficient, and environmentally friendly approach to mosquito control.
Figure 5. Developing process of RNAi yeast mosquitocides.
Part Collection for Autolytic Kill switch in E. coli
To address our potential end-users concerns towards biosafety of using live E. coli, we demonstrated several autolysis gene in E. coli for inducible kill switch setting, and thus make up a part collection including T4L(BBa_K5186018), Pa-T4L(BBa_K5186019), 2Pa-T4L(BBa_K5186020), X174E(BBa_K1835500). These parts are engineered to be IPTG-inducibly expressed and our tests show that gene E outperforms with the majority of cell lysis within one hour of induction. This makes gene E an optimal candidate for our kill switch component in the parts collection.
This collection serves as a valuable resource for the iGEM community and researchers for kill switch settings in E. coli and recovery of recombinant proteins or other intracellular products without the need for mechanical or chemical cell disruption methods.
Figure 6. Various autolytic genes expression cassettes are engineered for kill switch setting in E. coli strain DH5a. (a) Genetic circuit construction of strains KS 1-4. (b) Gel electrophoresis analysis of transformed autolytic genes expression cassettes. (c) Growth curve of control (E. coli strain DH5a) and strains KS 1-4.
Droplet dispenser module in device
To ensure the feasibility of our project and functionality of our product, we carefully examined feedback received from stakeholders. In feedback, we noticed that our stakeholders want us to ensure biosafety and minimize the risk of leakage of bacteria used in our hardware. With this consideration, we designed the hardware to fully enclose the living bacteria, and added a controllable automatic drip module to the device.
We've already opened sourced our Droplet dispenser module assembly and implementation methods. For details, check our our Hardware page. By adopting our kill switch mechanism, future iGEM teams and stakeholders can ensure safer and more responsible microbial management, promoting environmental safety.
Figure 7. Integration of Screw Motor, Syringe, and Serum tube
Method of Mosquito feeding experiment
Having confirmed the successful production of HMBPP in E. coli and various shRNAs designed against key mosquito survival genes in S. cerevisiae, we proceeded with mosquito feeding experiments with the approval of our check-in form.
To ensure safety and mitigate potential risks, we have established a comprehensive training instruction for our experimental team. The methodologies were provided by Jing Wu, a PhD candidate from Professor Chen Xiaoguang's research group.
Our documentation, including the check-in form and the mosquito feeding experiment methods, serve as a valuable reference for other iGEM teams undertaking similar mosquito-related research attempts.