1. Contribution Summary
Our team developed a yeast-based RNA interference (RNAi) testing platform that allows evaluation of short hairpin RNA (shRNA) efficiency without the use of animal or mammalian cells. The system employs an engineered strain of Saccharomyces cerevisiae (CEN.PK2-1C) with re-integrated RNAi machinery and a fluorescence reporter construct to visualize gene silencing activity. This tool enables other iGEM teams, especially high school teams working under biosafety or resource limitations, to rapidly screen RNAi constructs targeting specific genes.
2. Design
In compliance with iGEM safety rules, and particularly the restrictions placed on high school teams, animal experiments are strictly prohibited. To ensure animal welfare, we did not, and do not plan to, test shRNA efficiency in real animals.
In our project, we aim to solve the issue that many cat lovers are allergic to cat allergens secreted from salivary and sebaceous glands. These tissues are not available commercially, and existing feline cell lines (e.g., kidney-derived) require advanced biosafety-level culture facilities (dedicated clean cell culture rooms, HEPA-filtered incubators, and stringent animal cell culture equipment). Besides, preparation time of 2–3 months for cell line establishment, according to the cell line supplier and mammalian cell experts from our Human Practices interviews, were inaccessible with our lab resources and timeline.
Because Saccharomyces cerevisiae lacks a native RNA interference (RNAi) machinary—having lost the Dicer genes—it cannot normally be used for shRNA testing. Through our human practices work, we identified LINK-Spider, a synthetic biology unicorn offering a genetically engineered yeast strain (S. cerevisiae CEN.PK2-1C), in which the complete RNAi machinery has been reintroduced. This strain provided a safe, accessible, and scientifically rigorous chassis for testing shRNA activity.
For the reporter design, we selected mTurquoise, a cyan fluorescent protein variant with low spectral overlap with cellular autofluorescence. It is more sensitive than GFP for quantitative fluorescence assays. The mRNA sequences of the five cat allergen genes were fused at the C-terminus of the mTurquoise coding sequence. This configuration ensures that allergen-derived sequences do not interfere with protein folding or reporter expression, while still allowing RNAi-mediated silencing to be detected as a reduction in fluorescence intensity.
shRNA sequences can be expressed under the IPTG-inducible Ptac promoter in E. coli HT115, purified, and introduced into the engineered yeast chassis. The resulting fluorescence data confirmed that the yeast RNAi system could accurately detect gene silencing across multiple allergen targets, validating its reliability and reusability.
3. How Other Teams Can Use It
Future iGEM teams can adapt our RNAi platform to test their own RNA-based constructs:
Clone any target sequence downstream of mTurquoise in the yeast reporter plasmid.
Design and produce shRNA constructs using the same Ptac-shRNA expression backbone.
Transform the shRNA and reporter plasmids into the RNAi-enabled S. cerevisiae strain.
Measure fluorescence changes to determine knockdown efficiency.
This workflow provides a simple, reproducible framework for testing gene silencing designs in a biosafe environment, making RNAi experimentation accessible to all iGEM teams.
4. Documentation Added to Registry Parts
We added detailed functional data and experimental validation to our Registry Parts.
1) The usage and effects of all relevant shRNA constructs were documented in the part collection f
2) The fusion reporter with mTurquoise–allergen was also documented in the relevant parts page, and you can access these parts from the central collection hub . The documentations confirmed successful fluorescence in yeast and validated fluorescence reduction upon shRNA induction.
5. Brief summary of the Reporter System
To evaluate the knockdown efficiency of our designed shRNAs, we constructed a yeast reporter system expressing the cat allergen Fel d1 CH1 fused with mTurquoise. DNA sequence of Fel d1 CH1 mRNA was synthesized using a nucleic acid synthesizer by commercial suppliers. The Fel d1 CH1 sequence was amplified by PCR with overhangs and assembled into the pType 9K vector via Gibson assembly. The resulting constructs were transformed into E. coli DH5α, and sequencing confirmed the correct insertion. Verified plasmids were then isolated and transformed into S. cerevisiae CEN.PK2-1C. Fluorescence expression was confirmed under blue light illumination using a gel documentation system.
Figure 1. Design and construction of the mTurquoise-mRNA yeast reporter system.
Fluorescence was measured in yeast cells transformed with plasmids under induced (shRNA expressed) and uninduced (shRNA not expressed) conditions. To account for differences in cell density, values were normalized against OD600, yielding relative fluorescence intensity per cell.
Figure 2. Fluorescent protein expression in transformed yeast colonies. (A) P-Type9k (without cat allergen mRNA); (B) Type 9k-Fel d1-CH1; (C) Type 9k-Fel d1-CH2; (D) Type 9k-Fel d2; (E) Type 9k-Fel d4; (F) Type 9k-Fel d7
Figure 3. Knockdown efficiency of shRNA targeting Fel d1 CH1. Three shRNA were independently designed and tested. The fluorescence intensity of the mTurquoise reporter was normalized against the cell density OD600. Statistical significance from t-tests: *: p<0.05; ** p<0.01; ***: p<0.001.
The results demonstrate that all three shRNA constructs significantly suppressed Fel d1 CH1 expression compared with the uninduced controls (p < 0.05, p < 0.01, p < 0.001). The results also demonstrated the functionality of the mTurquoise-mRNA yeast reporter system.
