iGEM 2025 — Team bwya

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

Engineering

* [Overview](#p-1)
* [1.Engineering Cycle: Design](#p-2)
* [2.Engineering Cycle: Build](#p-3)
* [3.Engineering Cycle: Test](#p-4)
* [4.Engineering Cycle: Learn](#p-5)
* [REF](#p-6)

## 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

![image](https://static.igem.wiki/teams/5927/engineering/engineering1.webp)

**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**

![image](https://static.igem.wiki/teams/5927/engineering/engineering2.webp)

**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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering3.webp)

**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**

![image](https://static.igem.wiki/teams/5927/engineering/engineering4.webp)

**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]

![image](https://static.igem.wiki/teams/5927/engineering/engineering5.webp)

**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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering6.webp)

**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]

![image](https://static.igem.wiki/teams/5927/engineering/engineering7.webp)

**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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering8.webp)

**Figure** **8****. Knockdown efficiency of shRNA targeting Fel d1 CH****2****.** 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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering9.webp)

**Figure** **9****. Knockdown efficiency of shRNA targeting Fel d****2****.** 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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering10.webp)

**Figure** **10****. Knockdown efficiency of shRNA targeting Fel d****4****.** 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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering11.webp)

**Figure** **11****. Knockdown efficiency of shRNA targeting Fel d****7****.** 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.

- **Fel d1 CH1:** All three shRNAs (shRNA1–3) demonstrated activity. We will mix all three constructs and transform the yeast to see if knockdown is enhanced compared to single-construct performance.
- **Fel d1 CH2:** Only one shRNA (shRNA3) showed activity and thus we won’t test this case.
- **Fel d2:** All three shRNAs showed activity and similar efficiencies. The mixture includes all to assess potential additive effects.
- **Fel d4:** All three shRNAs showed activity, and shRNA3 is lower in efficiency. All three constructs are included in the test.
- **Fel d7:** Among the three constructs, shRNA1 and shRNA3 showed activity. Only these two are included in the mixture.

Some of the previous single-shRNA constructs are also included for comparison.

![image](https://static.igem.wiki/teams/5927/engineering/engineering/eng1.webp)

**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.

## REF

[1] Bonnet, B., Messaoudi, K., Jacquet, A., Moingeon, P., & Moussu, H. (2018). An update on molecular cat allergens: Fel d 1 and what else? \*Allergy, Asthma & Clinical Immunology\*, 14, 14. https://doi.org/10.1186/s13223-018-0239-8

[2] Sparkes, A. H. (2021). Human allergy to cats: A review for veterinarians on prevalence, causes, diagnosis and control. \*Journal of Feline Medicine and Surgery\*, 23(12), 1097–1111. https://doi.org/10.1177/1098612X211036793

[3] Cleveland, C. W., III, et al. (2024). Genetic diversity of the major cat allergen, Fel d 1. \*PNAS Nexus\*, 3(11), pgae447. https://doi.org/10.1093/pnasnexus/pgae447

[4] Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S., & Khvorova, A. (2004). Rational siRNA design for RNA interference. \*Nature Biotechnology\*, 22(3), 326–330. https://doi.org/10.1038/nbt936

[5] Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., & Tuschl, T. (2001). Duplexes of 21‑nucleotide RNAs mediate RNA interference in cultured mammalian cells. \*Nature\*, 411(6836), 494–498. https://doi.org/10.1038/35078107

[6] Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., & Ahringer, J. (2001). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in \*Caenorhabditis elegans\*. \*Genome Biology\*, 2(1), research0002. https://doi.org/10.1186/gb-2000-2-1-research0002

[7] de Boer, H. A., Comstock, L. J., & Vasser, M. (1983). The tac promoter: A functional hybrid derived from the trp and lac promoters. \*Proceedings of the National Academy of Sciences of the USA\*, 80(1), 21–25. https://doi.org/10.1073/pnas.80.1.21

[8] Cambray, G., Guimaraes, J. C., & Arkin, A. P. (2013). Measurement and modeling of intrinsic transcription terminators. \*Nucleic Acids Research\*, 41(9), 5139–5148. https://doi.org/10.1093/nar/gkt163

[9] Dabrowski, A. J., Falcioni, F., & Aarons, A. (1990). Cat skin as an important source of Fel d I allergen. \*Journal of Allergy and Clinical Immunology\*, 86(4), 462–465. https://doi.org/10.1016/S0091-6749(05)80200-3

[10] Drinnenberg, I. A., et al. (2009). RNAi in budding yeast. \*Science\*, 326(5952), 544–550. https://doi.org/10.1126/science.1176945

[11] Suk, K., Choi, J. H., Kim, J., & Jacobson, A. (2011). Reconstitution of human RNA interference in budding yeast. \*Nature Communications\*, 2, 101. https://doi.org/10.1038/ncomms1105

[12] Goedhart, J., et al. (2012). Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93% (mTurquoise2). \*Nature Communications\*, 3, 751. https://doi.org/10.1038/ncomms1738

[13] Gibson, D. G., et al. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. \*Nature Methods\*, 6(5), 343–345. https://doi.org/10.1038/nmeth.1318

[14] Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. \*Methods\*, 25(4), 402–408. https://doi.org/10.1006/meth.2001.1262

[15] Jackson, A. L., & Linsley, P. S. (2010). Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. \*Nature Reviews Drug Discovery\*, 9(1), 57–67. https://doi.org/10.1038/nrd3010

[16] Birmingham, A., et al. (2006). 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. \*Nature Methods\*, 3(3), 199–204. https://doi.org/10.1038/nmeth854

[17] Endy, D. (2005). Foundations for engineering biology. \*Nature\*, 438(7067), 449–453. https://doi.org/10.1038/nature04342

[18] iGEM Foundation. (2025). Animal Use Policy & Safety Policies. Retrieved from https://responsibility.igem.org/safety-policies/animal-use

[19] Sparkes, A. H., et al. (2012). ISFM guidelines on practical aspects of feline handling and nursing care. \*Journal of Feline Medicine and Surgery\*, 14(5), 337–349. https://doi.org/10.1177/1098612X12445021

[20] Moore, M. J., et al. (2015). Argonaute CLIP reveals determinants of miRNA target selection in mammals. \*Nature Structural & Molecular Biology\*, 22(2), 159–166. https://doi.org/10.1038/nsmb.2950

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

Experiments

[1.
Microbial Media Preparation and Cultivation 1](#_Toc32528)

[1.1 *E.
Coli* Media
Preparation 1](#_Toc22314)

[1.2 YeastMedia
Preparation 2](#_Toc1696)

[2.
Molecular cloning 3](#_Toc4766)

[2.1
The construction of yeast reporter 3](#_Toc6768)

[2.1.1
Plasmid Extraction 3](#_Toc20172)

[2.1.2
PCR
amplification 4](#_Toc8369)

[2.1.3
Electrophoresis 5](#_Toc9925)

[2.1.4
Gel extraction 6](#_Toc1846)

[2.1.5
Gibson Assembly 7](#_Toc7178)

[2.1.6
Transformation of *E.
coli*
DH5a 8](#_Toc20617)

[2.1.7
Transformation
of S. cerevisiae CEN. PK2-1C 9](#_Toc22983)

[2.2
The construction of ShRNA
expression cassette 11](#_Toc2762)

[2.2.1 T4
PNK Phosphorylation 11](#_Toc3768)

[2.2.2
Digestion 12](#_Toc1657)

[2.2.3
Extraction of Digestion Product 12](#_Toc31560)

[2.2.4
Ligation 13](#_Toc5268)

[2.2.6
Sequencing 13](#_Toc32181)

[2.2.7
Transformation of E. coli HT115 13](#_Toc3767)

[2.2.8
Expanding Cultivation & IPTG Induction 14](#_Toc29750)

[2.2.9
RNA Extraction 14](#_Toc16666)

[2.2.10
RNA Electrophoresis 16](#_Toc19834)

[3.Validation
of RNAi knockdown efficiency 16](#_Toc19875)

[3.1
RNA Chemical Transformation 16](#_Toc8699)

[3.2.
Quantify fluorescence
intensity 17](#_Toc6248)

1. # Microbial Media Preparation and Cultivation

1. ## *E. Coli* Media Preparation

**LB
Media**

| Component | Quantity |
| Peptone | 10 g |
| Yeast Extract | 5 g |
| NaCl | 10 g |
| ddH2O | 1000 mL |

Add
20 g Agar when preparing agar plates (solid medium)

Autoclave
in 121°C for 15 min.

Under
sterile conditions, supplement LB media with kanamycin (50 µg/mL) or
chloramphenicol (20 µg/mL) for selection.

Pour
the media into Petri dishes to solidify.

Store
both plates at 4°C until use.

2. ## YeastMedia Preparation

**YPD
Agar Media**

| Component | Quantity |
| Tryptone | 10 g |
| Yeast Extract | 10 g |
| Agar | 20 g |
| ddH2O | 950 mL |

Autoclave
in 121°C for 15 min.

Under
sterile conditions, add 50 mL of 40% (w/v) glucose solution to the
YPD medium to achieve a final concentration of 2% glucose.

Pour
the media into Petri dishes to solidify.

Store
plates at 4°C until use.

**SC-Ura
Media**

| Component | Quantity |
| Yeast Nitrogen Base (without amino acids) | 0.6 g |
| (NH4)2SO4 | 1.75 g |
| DO supplement-Ura | As stated in the product manual |
| ddH2O | 950 mL |

Add
20 g Agar when preparing agar plates (solid medium)

Autoclave
in 121°C for 15 min.

Under
sterile conditions, add 50 mL of 40% (w/v) glucose solution to the
medium to achieve a final concentration of 2% glucose.

Pour
the media into Petri dishes to solidify.

Store
plates at 4°C until use.

**40%
glucose (w/v) solution**

1.
Place a beaker containing 800 mL of distilled/deionized water on a
magnetic stirrer and begin stirring.

2.
While stirring, slowly add 400 g of glucose.

Caution:
Adding water to a large mass of glucose can cause severe clumping.
Always add the glucose to the water.

3.
Continue stirring until the glucose is completely dissolved.

4.
Transfer the solution to a 1 L graduated cylinder.

5.
Bring the final volume to 1 L by adding distilled/deionized water.

6.
Pass the fully dissolved solution through a 0.22μm filter and into a
fresh, sterile container.

2. # Molecular cloning

## 2.1 The construction of yeast reporter

### 2.1.1 Plasmid Extraction

We
used TIANpure
Mini Plasmid Kit
(DP104) for the plasmid extraction.

1. Column
   equilibration: Place a Spin Column CP3 in a clean collection tube,
   and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm
   (~13,400×g) in a table-top microcentrifuge. Discard the
   flowthrough, and set the Spin Column CP3 back into the collection
   tube.

2.
Harvest 5 ml bacterial cells in a microcentrifuge tube by
centrifugation at 4,000 rpm for 10 min at room temperature (15-30°C),
then remove all traces of supernatant by gently inverting the open
centrifuge tube until all medium has been drained.

3.
Resuspend pelleted bacterial cells in 250 μl Buffer P1
and transfer to 1.5 mL microcentrifuge
tubes.

4.
Add 250 μl Buffer P2 and invert the tube 6-8 times.

5.
Add 350 μl Buffer P3 and invert the tube 6-8 times.

6.
Centrifuge for 10 min at 12,000 rpm (~13,400×g)

7.
Carefully
transfer the supernatant from step 6 to the Spin Column CP3 by
decanting or pipetting. Centrifuge for 1 min at 12,000 rpm
(~13,400×g). Discard the flow-through and set the Spin Column CP3
back into the Collection Tube

8.
Wash the Spin Column CP3 by adding 600 µl Buffer PW (ensure the
ethanol (96%-100%) has been added to Buffer PW) and centrifuging for
30-60 sec at 12,000 rpm (~13,400×g). Discard the flow-through and
set the CP3 back into the Collection Tube.

9.
Wash Spin Column CP3 by adding 600 µl Buffer PW and centrifuging for
1 min at 12,000 rpm (~13,400×g).

10.
Discard the flow-through, and centrifuge for an additional 2 min at
12,000 rpm (~13,400×g) to remove residual wash Buffer PW.

11.
Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To
elute DNA, add 50-100 μl Buffer TB or water (pH7.0-8.5) to the
center of the Spin Column CP3, let it stand for 2 min, and then
centrifuge for 2 min at 12,000 rpm (~13,400×g)

### 2.1.2 PCR amplification

1.
Label
7 microcentrifuge tubes (See table)

2.
Add
25 μL mix, 2 μL forward primer, 2 μL reverse primer, 2 μL DNA
template, and 19 μL water to each tube.

| Fragment Name | DNA Template | Primers (F/R) | Length/bp | Annealing Temp/°C |
| Type9k-F1 | P-Type9k | Type9k-gib-F1 | 3353 | 56 |
| Type9k-gib-R1 |
| Type9k-F2 | P-Type9k | Type9k-gib-F2 | 2872 | 56 |
| Type9k-gib-R2 |
| F-Fel d1-CH1 | P-Fel d1-CH1 | d1CH1-gib-F | 481 | 56 |
|  |
| d1CH1-gib-R |
| F-Fel d1-CH2 | P-Fel d1-CH2 | d1CH2-gib-F | 534 | 56 |
| d1CH2-gib-R |
| F-Fel d2 | P-Fel d2 | d2-Gib-F | 1944 | 56 |
| d2-Gib-R |
| F-Fel d4 | P-Fel d4 | d4-Gib-F | 918 | 56 |
| d4-Gib-R |
| F-Fel d7 | P-Fel d7 | d7-Gib-F | 798 | 56 |
| d7-Gib-R |

2. Run
   PCR with the following settings:

### | Step | Temperature (°C) | Time | Cycles | | 1. Initial Denaturation | 95 | 5 min | 1 cycle | | 2. Denaturation | 95 | 30 s | Repeat steps 2-4 for 40 cycles | | 3. Annealing | 56 | 30 s | | | 4. Extension | 72 | X min (see table for details) | | | 5. Final Extension | 72 | 10 min | 1 cycle | | 6. Hold | 4 | Indefinite | 1 cycle |

### 2.1.3 Electrophoresis

| Materials | Quantity |
| 1xTAE Buffer | 40 mL |
| Agarose | 0.6 g |
| SYBR Gold | 4 μL |
| 6X Loading Buffer | 8 μL per PCR product tube |
| DNA Markers | DL 2000 |

1. Mix
   40
   mL 1xTAE
   Buffer with 0.6
   g agarose in a conical flask.
2. Heat
   in a microwave until dissolved.
3. Cool
   and
   add 4
   μL SYBR
   Gold mix
   well.
4. Pour
   into a gel casting tray, let solidify.
5. Add
   6X loading buffer to PCR products
6. Load
   samples into gel wells.
7. Load
   10 μL DL2000
   marker
8. Run
   electrophoresis at 180 V for 15 min.
9. Visualize
   the gel under UV light.

### 2.1.4 Gel extraction

We
use Universal DNA Purification Kit (GDP214) for gel extraction.

1.Column
equilibration: Add 500 μl Buffer BL to the Spin Column CB2 (in a 2
ml Collection Tube). Centrifuge at 12,000 rpm (~13,400 × g) for 1
min.

2.
Cut the target DNA band from the agarose gel and transfer it into a
clean centrifuge tube, and then weigh the gel block.

3.
Add equal volume of Buffer PC to the volume of gel.

4.
Incubate
at 50°C for about 10 min by inverting up and down the tube until the
agarose gel dissolved completely.

5.
Transfer the whole solution to the Spin Column CB2. Centrifuge at
12,000 rpm (~13,400 × g) for 1 min.

5.
Add 600 µl Buffer PW (Ensure that ethanol (96-100%) has been added
to Buffer PW before use) into the Spin Column CB2 and centrifuge at
12,000 rpm (~13,400 × g) for 1 min.

6.
Repeat Step 5.

7.
Set the Spin Column CB2 back to the Collection Tube and centrifuge
the column at 12,000 rpm (~13,400 × g) for an additional 2 min to
remove residual wash buffer. Discard the flow-through, and allow the
column to air dry with the cap open for 2-5 mins to dry the membrane.

8.
To elute DNA, place the column in a clean 1.5 ml microcentrifuge
tube.

Add
appropriate volume of Buffer EB (For fragments >4 kb, Buffer EB
should be preheated to 65-70°C) to the center of the membrane,
incubate at room temperature for 2 min, and centrifuge at 12,000 rpm
(~13,400 × g) for 2 min.

### 2.1.5 Gibson Assembly

Set
up the following reaction on ice:

| Type 9k-Fel d1-CH1 | |
| Materials | Usage |
| Gibson Assembly Mix | 5 ul |
| Type9k-F1 | 70 ng |
| Type9k-F2 | 65 ng |
| F-Fel d1-CH1 | 80 ng |
| dd H2O | Up to 10 ul |

| Type 9k-Fel d1-CH2 | |
| Materials | Usage |
| Gibson Assembly Mix | 5 ul |
| Type9k-F1 | 70 ng |
| Type9k-F2 | 65 ng |
| F-Fel d1-CH2 | 80 ng |
| dd H2O | Up to 10 ul |

| Type 9k-Fel d2 | |
| Materials | Usage |
| Gibson Assembly Mix | 5 ul |
| Type9k-F1 | 70 ng |
| Type9k-F2 | 65 ng |
| F-Fel d2 | 125 ng |
| dd H2O | Up to 10 ul |

| Type 9k-Fel d4 | |
| Materials | Usage |
| Gibson Assembly Mix | 5 ul |
| Type9k-F1 | 70 ng |
| Type9k-F2 | 65 ng |
| F-Fel d4 | 60 ng |
| dd H2O | Up to 10 ul |

| Type 9k-Fel d7 | |
| Materials | Usage |
| Gibson Assembly Mix | 5 ul |
| Type9k-F1 | 70 ng |
| Type9k-F2 | 65 ng |
| F-Fel d7 | 50 ng |
| dd H2O | Up to 10 ul |

Incubate
samples in a thermocycler at 50°C for 15 minutes.

Following
incubation, store samples on ice or at -20°C for subsequent
transformation.

### 2.1.6 Transformation of *E. coli* DH5a

1.Take
competent cells out of -80°C and thaw on ice (approximately 20-30
mins).

2.
Mix 5 μl of assembled
DNA into 50 μL of
competent cells.
GENTLY mix by flicking the bottom of the tube with your finger a few
times.

3.
Incubate the competent
cell/DNA mixture on ice for 20-30 mins.

4.
Heat shock each
transformation tube by placing the tube into a 42°C water bath for
45 secs.

5. Put the tubes back on ice for 2 min.
6. Add 500
   μl LB media (without antibiotic) to the bacteria and grow in 37°C
   shaking incubator for more
   than 45 min.
7. Centrifuge to
   pellet the cells and decant most of the media.
8. Resuspend the
   cells and plate on the selection media according to the plasmid
   transformed.
9. Incubate plates at 37°C overnight.

### 2.1.7 Transformation of S. cerevisiae CEN. PK2-1C

Stock
solution

| 1M LiAc | | |
| Materials | Usage | Note |
| LiAc-2H2O | 5.1 g/50 mL |  |

| 50% PEG3350 | | |
| Materials | Usage | Note |
| PEG3350 | 25 g/50 mL |  |

1. Inoculate
   5
   mL
   of liquid YPDand
   incubate with shaking overnight at 30
   °C
   .
2. Inoculate
   0.5
   mL overnight yeast cultured solution into
   **5****0****mL****2xYPD**.
3. Incubate
   the culture at 30°C on a shaker at 200 rpm until OD600~0.5-1.2.
   This will take around 4-5
   hours.
4. Harvest
   the culture in a sterile 50mL centrifuge tube at 4000
   rpm for 5 min.
5. Pour
   off the medium, resuspend the cells in 25mL of sterile water and
   centrifuge again.
6. Pour
   off water,
   resuspend the cells in 25mL of sterile water and centrifuge again.
7. Pour
   off the water,
   resuspend
   the bacterial pellet from step (6) with X mL (i.e., the number of
   competent cell aliquots to be prepared) of sterile water.
8. Distribute
   the resuspended solution into X sterile 1.5 mL microcentrifuge tubes
   (1 mL per tube).
9. Centrifuge
   the tubes using a microcentrifuge at room temperature and 5000 rpm
   for 5 minutes.
10. After
    centrifugation, carefully discard the supernatant to obtain the
    yeast competent cell pellet.

11.
Preparation of LiAc Mix

LiAc
Mix Recipe (Single Aliquot)

| LiAc Mix Recipe (each) | | |
| Materials | Usage | Note |
| 50% PEG3350 | 260 μL |  |
| 1 M LiAc | 36 μL |  |
| Sterile Water | 4 μL |  |
| 2 mg/mL ssDNA | 10 μL | Heat ssDNA at 95°for 5 min before use |
| dd H2O | Up to 10 ul |  |

For
n aliquots: Prepare a mixture with a total volume of (n+2) times the
single aliquot volume (i.e., scale up the single aliquot recipe by
(n+2) times). After preparation, aliquot the mixture into (n+2)
sterile 1.5 mL microcentrifuge tubes (including the required number
for experimental and control groups, plus two extra aliquots as
backups for potential loss).

(9)
Preparation of DNA Mix

Prepare
DNA Mix solutions for the experimental and control groups separately.
Ensure that each DNA mix contains at least 250 ng of plasmid DNA,
with a final volume of 50 μL.

(10)
Chemical Transformation

a.
Gently resuspend the competent cell pellet from step (7) with the
corresponding DNA mix (50 μL) (Note: Competent cells must be used
immediately after removal from the 4°C refrigerator).

b.
After resuspension, add 310 μL of the LiAc mix from step (8) to each
tube. Gently flick the tube or vortex to ensure thorough mixing,
forming the Transformation Mix.

c.
Precisely set a metal bath or water bath to 42°C. Place the
Transformation Mix tubes from step b into the bath and heat-shock for
40 minutes.

d.
After heat shock, centrifuge the tubes using a microcentrifuge at
room temperature and 5000 rpm for 2 minutes. Carefully aspirate and
discard the supernatant using a pipette, avoiding loss of the
bacterial pellet at the bottom of the tube.

e.
Add 500 μL of sterile water to each tube and gently pipette or
vortex to resuspend the bacterial pellet. After resuspension, pipette
100 μL of the bacterial suspension and spread it evenly onto SC-Ura
(Uracil-Deficient) selection plates.

f.
Invert the spread plates and place them in a 30°C incubator to
culture for 2-3 days. After cultivation, successfully transformed
yeast monoclonal colonies should be visible.

## 2.2 The construction of ShRNA expression cassette

### 2.2.1 T4 PNK Phosphorylation

**(1)
Preparation of Oligo Pool**

Mix
5 μL each of the upstream and downstream oligo DNA fragments. Vortex
to mix thoroughly, then briefly centrifuge to collect the solution at
the bottom of the tube to obtain the DNA Oligo Pool.

For
example: CH1-shRNA1-F and CH1-shRNA1-R, CH1-shRNA2-F and
CH1-shRNA2-R.

**(2)
T4 PNK Phosphorylation**

Prepare
the reaction mixture according to the table below, then place it in a
PCR machine and run the reaction program shown in Figure 1.

T4
PNK Phosphorylation reaction
mix (50
μL )

|  | 50 μL |
| --- | --- |
| 10 × T4 ligation buffer | 5 |
| DNA oligo pool | 1 |
| T4 Polynucleotide Kinase | 1 |
| dd H2O | 43 |

**T4
PNK Phosphorylation Reaction Program**

|  |  |  |
| --- | --- | --- |
| Step | Programme | Notes |
|  |  |  |
| --- | --- | --- |
| 1 | 37°C，1 h | Phosphorylate oligo DNA ends with T4 PNK |
| 2 | 98°C，5 min | Enzyme inactivation |
| 3 | 10°C，∞ |  |

### 2.2.2 Digestion

|  | 50μL |
| --- | --- |
| rCutSmart buffer | 5 |
| [XbaI](https://www.neb.com/en/products/r0145-xbai#!/_blank) | 3 |
| BsaI-HFv2 | 3 |
| pW1 | 1 μg |
| dd H2O | Up to 50 μL |

|  |  |  |
| --- | --- | --- |
| Step | Programme | Notes |
|  |  |  |
| --- | --- | --- |
| 1 | 37°C，30 min |  |
| 2 | 80°C，20 min | inactivation |
| 3 | 10°C，∞ |  |

### 2.2.3 Extraction of Digestion Product

Refer
to the instructions of the Universal DNA Purification Kit (GDP214).
(Section
2.1.4 Gel extraction)

###

### 2.2.4 Ligation

|  | **10****μL** |
| --- | --- |
| 10xT4 ligation Buffer | 1 |
| RD-pW1 | 60 ng |
| T4 PNK treated shRNAs oligo | 5 |
| T4 DNA ligase | 0.5 |
| dd H2O | Up to 10 μL |

**Transformation
of** ***E.
coli*** **DH5α****(see section 2.1.6 )**

### 2.2.6 Sequencing

Since
the fragment lengths of positive and negative colonies are similar,
it is recommended to directly pick a single colony and culture
it into liquid LB medium containing kanamycin
overnight. On the next day morning, we extracted the plasmids
following the procedures described in **2.1.1
Plasmid Extraction.**
Send to the sequencing facilities to get sequence information.

### 2.2.7 Transformation of E. coli HT115

Follow
the instructions of the HT115 competent cell product.

(https://www.maokangbio.com/productView.action?id=9458)

### 2.2.8 Expanding Cultivation & IPTG Induction

(1)
Inoculation

Pick
a single colony of HT115 cells containing the transformed plasmid
from the plate and inoculate it into a test tube containing liquid LB
medium with kanamycin (K antibiotic). Shake at 37°C overnight to
obtain the shRNA expression bacterial stock.

(2)
Expanding Cultivation

Inoculate
the bacterial stock at a 1:100 ratio into a 250 mL conical flask
containing 50 mL of liquid LB medium with kanamycin. Shake at 37°C,
200–300 rpm, for 3–4 hours (4 hours) until OD600 reaches
approximately 0.6.

(3)
Induction

Before
induction, take 1 mL of the sample as a pre-induction sample and
store it at 4°C for subsequent RNA extraction and electrophoresis
verification. Then add 1 M IPTG to a final concentration of 0.4 mM.
Shake at 37°C for 4 hours.

(4)
Bacterial Collection

Centrifuge
the pre-induction sample and post-induction bacterial culture at
12,000 rpm for 2 minutes. Discard the supernatant and collect the
bacterial pellet for RNA extraction.

### 2.2.9 RNA Extraction

We
use RNAprep
Pure Cell/Bacteria Kit
(GDP-430) to extract RNAs.

1.
Collect bacteria by centrifuging for 12,000 rpm (~13,400 × g) at 4°C
in a
centrifuge
tube. Carefully remove all supernatant by aspiration, and proceed to
step 2. The
following
steps should be completed at RT (15-30°C).

3.
Re-suspend bacteria thoroughly with 100 μl TE buffer containing
lysozyme (recipe and incubation time have been showed as follow).

G-
bacterium 400 μg /ml
3-5
min

G+
bacterium 3 mg/ml
5-10
min

3. Add
   350 μl Buffer RL (Ensure that β-Mercaptoethanol has been added to
   Buffer RL before use). Vortex or pipet to mix, and ensure that no
   cell clumps are visible before proceeding to step
   5.
4. If
   insoluble precipitate has been formed, centrifuge for 2 min at
   12,000 rpm (~13,400 × g). Transfer supernatant to another
   centrifuge tubes.

5.
Add 250 μl ethanol to the cleared lysate, mix immediately by
pipetting. Transfer the sample, including any precipitate that may
have formed, to RNase-Free Spin Column CR3 placed in a 2 ml
RNase-Free Collection
Tube.
Close the lid gently, and centrifuge for 30-60 s at 12,000 rpm
(~13,400 × g). Discard the flow-through, and put the spin column
back to the Collection Tube.

6.
Add 350 μl Buffer RW1 to the RNase-Free Spin Column CR3. Close the
lid gently, and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g).
Discard the flow-through.

7.
Preparation of DNase I working solution: Add 10 μl DNase I stock
solution (see Preparation of DNase I stock solution) to 70 μl Buffer
RDD. Inverting the tube gently to mix.

8.
Add the DNase I working solution (80 μl) directly to the center of
RNaseFree Spin Column CR3, and place on the bench top for 15 min.

9.
Add 350 μl Buffer RW1 to the RNase-Free Spin Column CR3. Close the
lid gently, and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g).
Discard the flow-through.

10.
Add 500 μl Buffer RW to RNase-Free Spin Column CR3 (Ensure that
ethanol has been added to Buffer RW before use). Close the lid
gently, place in room temperature for 2 min and centrifuge for 30-60
s at 12,000 rpm (~13,400 × g). Discard the flow-through.

11.
Repeat step 10.

12.
Centrifuge at 12,000 rpm (~ 13,400 × g) for 2 min, and then discard
the waste liquid. Place the RNase-Free Spin Column CR3 at room
temperature for 2-5 mins to completely dry the residual washing
buffer in the column.

13.
Place the RNase-Free Spin Column CR3 in a new 1.5 ml RNase-Free
Collection Tube (supplied). Add 30-100 μl RNase-Free ddH2O directly
to the spin column membrane. Close the lid gently, place at room
temperature (15-30°C) for 2 min and then centrifuge for 2 min at
12,000 rpm (~13,400 × g) to elute the RNA.

14.
Purified
RNA is recommended be stored at -70°C.

### 2.2.10 RNA Electrophoresis

RNA
electrophoresis can be performed using the common 1% Agarose-TAE gel
method for DNA electrophoresis. Recommendations:

1.
Clean the electrophoresis tank thoroughly before use, replace with
fresh TAE electrophoresis buffer, and rinse related equipment with
DEPC water before use.

2.
If the RNA concentration meets the above standard of 100 μg/μL,
directly take 2 μL RNA sample and mix with 10 μL 6× loading
buffer. Load 3 μL of the mixture and run electrophoresis at 140 V
for 20 minutes for verification.

3.
After verification, store the extracted RNA samples at -20°C.

# 3.Validation of RNAi knockdown efficiency

## 3.1 RNA Chemical Transformation

1.
Inoculate
a
reporter yeast colony into a test tube containing
5 mL
SC-Ura liquid medium. Incubate at 30°C with shaking at 200 rpm
overnight.

2.
Inoculate
the overnight reporter yeast culture at a 5% ratio into a 250 mL
conical flask containing 50 mL of SC-Ura (-Uracil) liquid medium.
Incubate at 30°C with shaking at 200 rpm until OD600 reaches
~0.5-0.8.

3.Centrifuge
at 4,000 rpm, 20°C for 5 minutes, and discard the supernatant.

4.Resuspend
the cell pellet in 1/2 volume of DEPC water. Centrifuge at 4,000 rpm,
20°C for 5 minutes to complete one wash cycle.

5.Repeat
the washing step twice.

6.Take
the yeast competent cells and resuspend them in the corresponding RNA
mix. After resuspension, add 310 μL of RNA mix to each tube, mix
well, and incubate in a 42°C water bath for 40 minutes.

**RNA
Mix Composition**

| LiAc Mix Recipe (each) | | |
| Materials | Usage | Note |
| 50% PEG3350 | 260 μL |  |
| 1 M LiAc | 36 μL |  |
| RNA | ~10 μL | (10 µg) |
| 2 mg/mL ssDNA | 10 μL | Heat ssDNA at 95°for 5 min before use |
| Nuclease free H2O | Up to 310 ul |  |

7.After
the water bath incubation, centrifuge at 5,000 rpm for 2 minutes at
room temperature and discard the supernatant.

8.Resuspend
the cells in 500 μL of SC-Ura medium and transfer to a 2 mL culture
tube.

9.Incubate
at 30°C with shaking at 200 rpm for 16 hours for recovery.

#####

## 3.2. Quantify fluorescence intensity

Measure
fluorescence using a microplate reader (excitation wavelength: 488
nm, emission wavelength: 509 nm).

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

Results

* [Overview](#p-1)
* [1.Engineering Cycle: Design](#p-2)
* [2.Engineering Cycle: Build](#p-3)
* [3.Engineering Cycle: Test](#p-4)
* [4.Engineering Cycle: Learn](#p-5)
* [REF](#p-6)

## 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

![image](https://static.igem.wiki/teams/5927/engineering/engineering1.webp)

**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**

![image](https://static.igem.wiki/teams/5927/engineering/engineering2.webp)

**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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering3.webp)

**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**

![image](https://static.igem.wiki/teams/5927/engineering/engineering4.webp)

**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]

![image](https://static.igem.wiki/teams/5927/engineering/engineering5.webp)

**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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering6.webp)

**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]

![image](https://static.igem.wiki/teams/5927/engineering/engineering7.webp)

**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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering8.webp)

**Figure** **8****. Knockdown efficiency of shRNA targeting Fel d1 CH****2****.** 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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering9.webp)

**Figure** **9****. Knockdown efficiency of shRNA targeting Fel d****2****.** 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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering10.webp)

**Figure** **10****. Knockdown efficiency of shRNA targeting Fel d****4****.** 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.

![image](https://static.igem.wiki/teams/5927/engineering/engineering11.webp)

**Figure** **11****. Knockdown efficiency of shRNA targeting Fel d****7****.** 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.

- **Fel d1 CH1:** All three shRNAs (shRNA1–3) demonstrated activity. We will mix all three constructs and transform the yeast to see if knockdown is enhanced compared to single-construct performance.
- **Fel d1 CH2:** Only one shRNA (shRNA3) showed activity and thus we won’t test this case.
- **Fel d2:** All three shRNAs showed activity and similar efficiencies. The mixture includes all to assess potential additive effects.
- **Fel d4:** All three shRNAs showed activity, and shRNA3 is lower in efficiency. All three constructs are included in the test.
- **Fel d7:** Among the three constructs, shRNA1 and shRNA3 showed activity. Only these two are included in the mixture.

Some of the previous single-shRNA constructs are also included for comparison.

![image](https://static.igem.wiki/teams/5927/engineering/engineering/eng1.webp)

**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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