Overview
RNA Strategy: Millions of cat lovers suffer from allergies triggered by specific proteins in cat saliva, skin, and dander, such as Fel d 1-CH1, Fel d 1-CH2, Fel d 2, Fel d 4, and Fel d 7 [1, 2]. Conventional treatments like antihistamines do not target the allergens directly and often cause side effects. In contrast, RNA interference (RNAi) can specifically silence allergen-encoding mRNAs, thereby reducing the production of the allergenic proteins. In this project, we designed 15 shRNA sequences targeting five major allergens, amplified them in E. coli DH5α, and produced them in E. coli HT115. To evaluate their silencing efficiency, we constructed a yeast-based fluorescence reporter system in which shRNA activity can be quantified through reduction in reporter fluorescence. [1] [4] [5]
Yeast Reporter: The reporter system is especially worth mentioning. In accordance with iGEM high school rules, animal experiments were strictly prohibited, and feline cell lines were not available within a feasible timeframe. We employed a specially engineered yeast chassis, S. cerevisiae CEN.PK2-1C, with functional RNAi machinery as our reporter system. Beyond our project, this approach provides a broadly applicable platform for other iGEM teams to test animal-related gene circuits under resource-limited conditions.
Vision and Passion: We hope create a proactive, long-term allergy relief approach. This project also holds potential beyond cat allergies, with the RNAi platform offering a versatile approach for targeting a variety of environmental allergens, transforming allergy care with precision and innovation.
1. Engineering Cycle: Design
Figure 1. Workflow of the project design and functional testing. (1) Fifteen (15) shRNA sequences were designed and cloned into E. coli DH5α for amplification, then transferred into E. coli HT115 for shRNA production. (2) A yeast reporter system was constructed, using an engineered strain with RNAi machinery, expressing an mTurquoise reporter with five (5) Fel d n target mRNAs. (3) shRNA constructs were transformed into the yeast reporter, and fluorescence quenching was measured to assess shRNA silencing efficiency.
1.1: Design and production of shRNA constructs
We selected five allergen-derived targets (Fel d1–d7) [3] and designed three shRNA sequences for each, following established criteria for shRNA design, including appropriate stem–loop structures, GC content, and avoidance of off-target effects [4]. For the exact sequences of the shRNA constructs, you can refer to our Parts page [link]. [5]
For shRNA production, we used E. coli HT115, a strain deficient in RNase III, which prevents degradation of double-stranded RNA and thereby improves yield and stability of shRNA products [5]. Expression was driven by the IPTG-inducible Ptac promoter, terminated by a strong double terminator. The pair is widely used in synthetic biology for reliable induction and transcription for RNA related production [6]. [7] [8]
1.2: Construction of the reporter system: Compliance with iGEM rules and a yeast strategy
In compliance with iGEM safety rules, and particularly the restrictions placed on high school teams, animal experiments are strictly prohibited. [7] To ensure animal welfare, we did not, and do not plan to, test shRNA efficiency in real animals. [19]
The Felis domesticus allergens (Fel d1–Fel d7) are naturally produced in specialized cat secretory tissues, e.g., Fel d1 is primarily expressed in salivary and sebaceous glands [8], as well as in epithelial cells lining hair follicles [9]. These tissues are not available as commercial cell lines, 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 during evolution—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, throughout our wiki materials, we will simply refer to this specific strain as “yeast”) in which the complete RNAi machinery has been reintroduced. This strain provided a safe, accessible, and scientifically rigorous chassis for testing shRNA activity. [10] [11]
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. [12]
1.3: Functional testing of shRNA
Finally, shRNA produced by E. coli HT115 will be chemically transformed into the engineered yeast reporter strain. When the shRNA successfully binds and guides the RNAi machinery, the mTurquoise–allergen fusion mRNA is degraded, leading to silencing of the reporter and a measurable reduction in fluorescence intensity.
2. Engineering Cycle: Build
2.1 shRNA expressing systems
Figure 2. Gene circuit design and confirmation of our Fel d1 CH1-ShRNA1 construct. Other shRNA construct are designed and confirmed in similar ways.
To tackle cat allergies, we designed a series of short hairpin RNAs (shRNAs) targeting 5 major cat allergens: Fel d1 (CH1 and CH2 domains), Fel d2, Fel d4, and Fel d7. Each shRNA follows the same design principle: a sense strand matching the allergen sequence, a loop that allows the RNA to fold back on itself, and an antisense strand that pairs with the sense strand to form the hairpin structure. These constructs are placed under the control of the pTac promoter and terminated with a strong double terminator to ensure stable expression. Once expressed inside cells, the shRNAs are processed into siRNAs that can specifically silence allergen genes, potentially reducing allergen production and easing allergic reactions.
All shRNA sequences were commercially synthesized. The forward and reverse oligonucleotides were annealed to form double-stranded fragments, then phosphorylated using T4 polynucleotide kinase (T4 PNK). These phosphorylated inserts were cloned into the pW1 vector through double digestion with XbaI and BsaI-HFv2. Recombinant plasmids were transformed into E. coli DH5α, and positive clones were verified by sequencing.
Here we demonstrate the Fel d1 CH1 shRNA construct as an example. The schematic (top) shows the sense, loop, and antisense regions, with the corresponding nucleotide sequence displayed below. The sequence map (bottom) confirms that the Fel d1 CH1-ShRNA1 cassette was correctly cloned into the plasmid backbone. Sanger sequencing alignment and chromatogram trace verified that the construct was assembled without mutations. For clarity, we present the sequence and sequencing results for one representative shRNA; full details for all constructs are provided on our Parts page.
Figure 3. RNA gel electrophoresis confirming IPTG-inducible expression of Fel d shRNAs. U: uninduced. I: induced.
Plasmids containing the verified inserts were then isolated and transformed into E. coli HT115. Expression of the shRNA was induced with IPTG at a working concentration of 0.5mM and produced at 30 C overnight.
To validate transcription of our allergen-targeting constructs, we extracted total RNA from bacterial cultures. Each culture was grown under uninduced (U) or IPTG-induced (I) conditions, and RNA was analyzed by gel electrophoresis. The RNA marker (right) was used to estimate transcript size. A distinct band appears in the induced lanes at approximately 100 bp, consistent with the expected size of our shRNA transcripts. In contrast, the uninduced samples show little or no small RNA signal, demonstrating that expression is tightly controlled by the IPTG-inducible pTac promoter. These results confirm that our designed constructs are capable of producing the intended shRNAs. [7]
2.2 Building the reporter system
Figure 4. Target gene PCR amplification. Through PCR, we successfully obtained the target genes of the allergens with overhangs (Fel d1 CH1, Fel d1 CH2, Fel d2, Fel d4, and Fel d7). We also obtained sequences of the pType 9K vector.
PCR is used to amplify vector backbones and fragments for constructing reporter systems. Agarose gel electrophoresis was used to verify the size of PCR-amplified DNA fragments. The analysis confirmed all of the anticipated backbones and fragments appear in the proper bands. These verified products will subsequently be purified and assembled into the desired plasmids for the reporter system using Gibson Assembly®. [13]
Figure 5. PCR of various assembled reporter constructs showing successful reporter+mRNA. We selected mTurquoise as our reporter.
Single colonies from each LB agar plate were selected and subjected to PCR amplification. Multiple colonies were tested per plate to minimize the impact of potential errors and to ensure reliable verification. The PCR products from colonies on five different plates were then analyzed by agarose gel electrophoresis. The confirmed colonies showing the correct PCR band sizes were selected and cultured overnight for further experimentation.
Figure 6. 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
Yeast (S. cerevisiae CEN.PK2-1C) cells were transformed with plasmids carrying fluorescent protein genes and plated on selective medium. Colonies were imaged under UV illumination to visualize fluorescence. Panels A–F represent independent transformation plates of different constructs with varying fluorescent intensities. Bright green colonies indicate successful plasmid uptake and expression of the fluorescent protein, while variation in colony density and fluorescence intensity reflects differences in transformation frequency and plasmid expression levels.
3. Engineering Cycle: Test
To evaluate the knockdown efficiency of our designed shRNAs, we quantitatively monitored the fluorescence intensity in the yeast reporters. For each target of cat allergens, three different shRNAs (Fel d1 CH1-shRNA1, shRNA2, and shRNA3) were tested. 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. [14]
Figure 7. 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, shown in Fig. 7, 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). Among them, Fel d1 CH1-shRNA2 [Part number] exhibited the strongest knockdown efficiency, achieving the largest reduction in fluorescence. This indicates that shRNA2 is the most effective design for silencing Fel d1 CH1 in this system.
Figure 8. Knockdown efficiency of shRNA targeting Fel d1 CH2. 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, shown in the figure, indicate that among the three designed constructs, only Fel d1 CH2-shRNA3 [Part number] significantly suppressed Fel d1 CH2 expression compared with the uninduced control (p < 0.001). In contrast, Fel d1 CH2-shRNA1 and Fel d1 CH2-shRNA2 showed no significant effect, as the fluorescence intensities remained unchanged between the induced and uninduced samples. These findings suggest that shRNA3 is the most effective design for targeting Fel d1 CH2, whereas the other two constructs did not achieve detectable knockdown under the tested conditions.
Figure 9. Knockdown efficiency of shRNA targeting Fel d2. 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, shown in the figure, demonstrate that all three shRNA [Part number] constructs significantly reduced Fel d2 expression compared with the uninduced controls (p < 0.05). While the degree of suppression varied among the three designs, each construct achieved measurable knockdown of Fel d2, indicating that multiple shRNA sequences are capable of effectively targeting this allergen.
Figure 10. Knockdown efficiency of shRNA targeting Fel d4. 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, shown in the figure, demonstrate that all three shRNA constructs [Part number] significantly suppressed Fel d4 expression compared with the uninduced controls (p < 0.01). While the degree of suppression varied among the three designs, each construct achieved measurable knockdown, indicating that multiple shRNA sequences are capable of effectively targeting this allergen.
Figure 11. Knockdown efficiency of shRNA targeting Fel d7. 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, shown in the figure, demonstrate that shRNA1 and shRNA3 [Part number] significantly suppressed Fel d7 expression compared with the uninduced controls (p < 0.01 p<0.001). However, shRNA2 showed no significant effect, as the fluorescence intensities remained unchanged between the induced and uninduced samples.
4. Engineering Cycle: Learn
4.1 Experimental Investigation Summary
From our results, we observed that 12 out of the 15 designed shRNA constructs showed measurable silencing activity in the yeast reporter system, though the efficiency varied across targets. For example, all three shRNAs against Fel d1-CH1 were effective, while only one of the three targeting Fel d1-CH2 showed significant silencing. This indicates some of the targets might be very difficult to taget
4.2 Future Work 1: Regarding shRNA specificity
Our current assays measured efficiency but not specificity. In theory, we conducted BLAST and ensured the designed shRNA only specifically interact with their designed target. Nonetheless, wet lab results verification would be desired. To gain a deeper understanding, future work would test whether each shRNA acts specifically on its intended target. One strategy is to introduce shRNAs designed for Fel d1-CH1 into Fel d2 or Fel d4 reporter strains to demonstrate true specificity.
4.3 Future Work 2: RNA Delivery
Beyond specificity, further experimental designs should include exploring chemical modifications and delivery methods. Modified shRNAs and optimized loop structures could improve stability and reduce off-target risks. Delivery strategies such as direct shRNA transfection versus plasmid-based shRNA expression could also be compared in yeast as a model platform.
4.4 Regarding Cat Cell Lines
In the longer term, feline-derived cells could provide a more biologically relevant system. Currently, only kidney-derived cat cell lines are commercially available, and using them would require specialized biosafety culture facilities and longer timelines. To test shRNA function in such cells, Fel d gene expression constructs with fluorescent or luminescent reporters could be introduced, followed by analysis with fluorescence assays, qPCR, or transcriptome profiling. This would allow both validation of silencing efficiency and feedback for improved shRNA design. Unfortunately, such cell culture work lies beyond the resources and timeline of a high school team.
4.5 Regarding Animal Studies
If this strategy were ever to progress toward real-world application, animal studies in cats would be essential to assess both efficacy and safety. Such experiments fall outside iGEM rules and were not pursued in this project, in order to protect animal welfare.
4.6 Regarding Broader Safety Issues
Safety also remains a key consideration. We addressed potential off-target effects by performing BLAST searches, confirming that our designed shRNAs do not significantly match unintended feline or human transcripts. While this preliminary check suggests low risk, more rigorous genome-wide specificity analyses (e.g., RNA-seq or Argonaute pull-down) would be required in future studies. [15] [20]
4.7 Experiment Exploration in Progress
To improve silencing efficiency, we also plan to try combined-shRNA knockdown by transforming yeast with a mixture of multiple shRNAs targeting the same allergen gene. Since each construct targets a distinct region on the mRNA, combining them may enable more transcript degradation and stronger suppression of allergen expression.
Some of the previous single-shRNA constructs are also included for comparison.
Figure 12 Knockdown efficiency of shRNA combinations.
These combinatorial assays are currently in progress. Fluorescence measurement is still pending, but our yeast colonies have been successfully transformed and are growing on selective media. Results from this ongoing work will be presented at Jamboree.
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