iGEM 2025 — Team kyoto

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## Lab Notebooks

##### We provide our lab notebooks in PDF format.

* 1. Notebook1[Download](https://static.igem.wiki/teams/5910/notebook/labnote1kaitei.pdf)
* 2. Notebook2[Download](https://static.igem.wiki/teams/5910/notebook/labnote2kaitei.pdf)
* 3. Notebook3[Download](https://static.igem.wiki/teams/5910/notebook/labnote3.pdf)

##### You can also take a look at our lab notebooks in the browser below (it is recommended to browse them with PC).

## Notebook1

## Notebook2

## Notebook3

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Initial Plan

Cycle1

[Design](#tab1-cycle1-design)
[Build](#tab1-cycle1-build)
[Test](#tab1-cycle1-test)
[Learn](#tab1-cycle1-learn)

Cycle2

[Design](#tab1-cycle2-design)
[Build](#tab1-cycle2-build)
[Test](#tab1-cycle2-test)
[Learn](#tab1-cycle2-learn)

Cycle3

[Design](#tab1-cycle3-design)
[Build](#tab1-cycle3-build)
[Test](#tab1-cycle3-test)
[Learn](#tab1-cycle3-learn)

Cycle4

[Design](#tab1-cycle4-design)
[Build](#tab1-cycle4-build)
[Test](#tab1-cycle4-test)
[Learn](#tab1-cycle4-learn)

References

![](https://static.igem.wiki/teams/5910/eng/engineering.webp)

Initial Plan

Cycle1

[Design](#tab2-cycle1-design)
[Build](#tab2-cycle1-build)
[Test](#tab2-cycle1-test)
[Learn](#tab2-cycle1-learn)

Cycle2

[Design](#tab2-cycle2-design)
[Build](#tab2-cycle2-build)
[Test](#tab2-cycle2-test)
[Learn](#tab2-cycle2-learn)

Cycle3

[Design](#tab2-cycle3-design)
[Build](#tab2-cycle3-build)
[Test](#tab2-cycle3-test)
[Learn](#tab2-cycle3-learn)

References

![](https://static.igem.wiki/teams/5910/eng/engineering.webp)

Initial Plan

Cycle1

[Design](#tab3-cycle1-design)
[Build](#tab3-cycle1-build)
[Test](#tab3-cycle1-test)
[Learn](#tab3-cycle1-learn)

Cycle2

[Design](#tab3-cycle2-design)
[Build](#tab3-cycle2-build)
[Test](#tab3-cycle2-test)
[Learn](#tab3-cycle2-learn)

Cycle3

[Design](#tab3-cycle3-design)
[Build](#tab3-cycle3-build)
[Test](#tab3-cycle3-test)
[Learn](#tab3-cycle3-learn)

References

![](https://static.igem.wiki/teams/5910/eng/engineering.webp)

Overview

Viral Transmission Modeling

[Design](#tab4-cycle1-design)
[Build](#tab4-cycle1-build)
[Test](#tab4-cycle1-test)
[Learn](#tab4-cycle1-learn)

Protein Optimization

[Design](#tab4-cycle2-design)
[Build](#tab4-cycle2-build)
[Test](#tab4-cycle2-test)
[Learn](#tab4-cycle2-learn)

[Design](#tab4-cycle3-design)
[Build](#tab4-cycle3-build)
[Test](#tab4-cycle3-test)
[Learn](#tab4-cycle3-learn)

![](https://static.igem.wiki/teams/5910/eng/engineering.webp)

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dsRNA sensor
Apoptosis Inducer
Fusion Protein
Model Selection

# Wet Lab Cycle

# Creation and Characterization of a dsRNA Recognition Module

Initial Plan

##### We decided to use a truncated form of full-length RIG-I, in which the two CARD domains in N-terminal were removed, as a dsRNA-binding domain. Our goal is to create genetically engineered cells that express COCCO. However, during the development process, it was necessary to produce recombinant proteins to validate the function of individual parts. Specifically, to verify whether this domain binds to dsRNA as expected, we conducted in vitro assays in which purified recombinant protein was incubated with synthetic dsRNA. The protein in question is a long polypeptide of 85.6 kDa, and making it amenable to recombinant purification required multiple iterations of an engineering cycle.

Cycle1

#### Design

##### We initially planned to simply clone the corresponding region from the RIG-I cDNA and insert it into an expression vector, using Escherichia coli as the expression host.

#### Build

##### We designed a plasmid by inserting the sequence encoding an N-terminally His-tagged RIG-I-CARDapaf1 into the pET21(+) vector.

![](https://static.igem.wiki/teams/5910/eng/eng-fig1.avif)

##### *Figure 1. Domain diagram of His-RIG-I-CARDapaf1*

#### Test

##### During a discussion with Dr. Fujita as part of our Human Practices activities, we learned that expressing and purifying RIG-I in E. coli is technically challenging. As a result, researchers often use alternative expression systems such as insect cells.

#### Learn

##### We realized that using standard methods to express RIG-I in E. coli made purification difficult, and that a modified approach would be required to obtain functional protein.

Cycle2

#### Design

##### To improve the expression and solubility of RIG-I-CARDapaf1 in E. coli, we searched for prior research and found that fusion with solubility-enhancing tags such as SUMO could be effective. Based on this, we redesigned the plasmid by adding an N-terminal SUMO tag to the construct.

![](https://static.igem.wiki/teams/5910/eng/eng-fig2.avif)

##### *Figure 2. Domain diagram of SUMO-tagged RIG-I-CARDapaf1*

#### Build

##### We constructed the redesigned plasmid and transformed it into E. coli for protein expression and purification.

#### Test

##### We assessed protein expression and purification by SDS-PAGE. As a result, we were able to purify a small but detectable amount of SUMO-tagged RIG-I-CARDapaf1.

![](https://static.igem.wiki/teams/5910/eng/eng-fig3.avif)

##### *Figure 3. SDS-PAGE of purified RIG-I-CARDapaf1.*

#### Learn

##### We learned that appending a SUMO tag to the N-terminus of RIG-I-CARDapaf1 improves its solubility and allows partial purification in E. coli. This confirmed the utility of fusion tags in improving expression of difficult-to-purify proteins.

Cycle3

#### Design

##### To evaluate the dsRNA-binding properties of recombinant RIG-I and its mutants, we needed to prepare a well-defined double-stranded RNA (dsRNA) substrate. Although many studies on RIG-I use synthetic analogs like poly(I:C), such molecules are unsuitable for assays requiring sequence-specific quantification—such as pull-down assays followed by qRT-PCR. Therefore, inspired by prior studies [e.g., Cell (2019), https://doi.org/10.1016/j.cell.2019.03.004], we designed a method to synthesize dsRNA in vitro from a DNA template using the MEGAscript® T7 Transcription Kit.

![](https://static.igem.wiki/teams/5910/eng/eng-fig4.avif)

##### *Figure 4. DNA templates used for in vitro transcription.*

##### Two templates with different T7 promoter placements were designed to produce complementary single-stranded RNAs.

#### Build

##### We ordered DNA fragments (gBlocks) from IDT, designed with opposing T7 promoters to transcribe complementary RNA strands. These templates were then used with the MEGAscript® Kit to synthesize RNA strands. We transcribed two complementary ssRNAs simultaneously in a single tube, allowing them to anneal naturally during the transcription reaction and form dsRNA.

#### Test

##### We transcribed RNA from each template and purified the products using the PureLink® RNA Mini Kit. The purified RNAs were analyzed by polyacrylamide gel electrophoresis (PAGE) to confirm successful formation of dsRNA.

![](https://static.igem.wiki/teams/5910/eng/eng-fig5.avif)

##### *Figure 5. Gel electrophoresis of synthesized dsRNA showing the expected size band.*

#### Learn

##### We successfully synthesized dsRNA of the expected length and confirmed its formation through PAGE. This provided us with a suitable reagent for downstream functional assays, such as verifying RNA binding by the recombinant RIG-I fragments.

Cycle4

#### Design

##### To investigate whether the purified RIG-I-CARDapaf1 protein could bind to dsRNA in vitro, we planned a pull-down assay. The experiment involved incubating purified His-tagged RIG-I-CARDapaf1 with in vitro-transcribed dsRNA in binding buffer, then using nickel beads to capture the His-tagged protein along with any bound RNA. We aimed to detect and quantify the pulled-down dsRNA to assess binding efficiency.

#### Build

##### To validate binding specificity, we prepared three experimental conditions: no RIG-I-apaf1, with RIG-I-apaf1, and with a mutant RIG-I-apaf1. The RNA pulled down under each condition was then purified for further analysis.

#### Test

##### We quantified the amount of RNA recovered from each condition using qRT-PCR. The percent recovery was calculated relative to the initial input RNA. Each condition was performed in triplicate (n = 3), and results were statistically analyzed.

![](https://static.igem.wiki/teams/5910/eng/eng-fig6.avif)

##### *Figure 6. Results of the dsRNA pull-down assay using RIG-I-CARDapaf1.*

#### Learn

##### Surprisingly, a substantial amount of dsRNA was recovered even in the no-protein control, indicating strong non-specific binding to the nickel beads. As a result, we could not clearly distinguish between protein-dependent and background binding. We suspect that insufficient washing steps during bead handling contributed to the high background. Moving forward, we plan to optimize the washing conditions and potentially use alternative affinity tags or blocking agents to reduce non-specific binding. With these improvements, we anticipate that dsRNA binding by RIG-I-CARDapaf1 can be visualized more clearly in future assays.

References

* [1]Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. *Science*, *360*(6387), 436-439.

  https://www.cell.com/cell/fulltext/S0092-8674(19)30279-X
* [2]Cadena et al. (2019), Ubiquitin-Dependent and -Independent Roles of E3 Ligase RIPLET in Innate Immunity

  <https://doi.org/10.1016/j.cell.2019.03.017>

# Apoptosis Inducing Module

Initial Plan

##### COCCO is a chimeric protein designed by replacing the CARD domain of RIG-I (CARDrigI) with the CARD domain of Apaf1 (CARDapaf1). This modification is intended to enable COCCO to interact with the CARD domain of Caspase-9 (CARDcasp9), thereby triggering apoptosis. To validate this interaction, we planned to purify CARDcasp9 and test its binding with the RIG-I-CARDapaf1 construct previously described in our experiments. Specifically, we designed a pull-down assay in which bead-immobilized CARDcasp9 would be used to capture RIG-I-CARDapaf1 from solution.

Cycle1

#### Design

##### To enable CARDcasp9 to bind to Glutathione Sepharose, we designed GST-CARDcasp9, which features a GST-tag sequence from pGEX-6p-1 at its N-terminus. For expression in E. coli, the codon-optimized nucleotide sequence for GST-CARDcasp9 was inserted into the pET21(+) vector.

#### Build

##### The plasmid was synthesized by Twist Bioscience. We transformed the prepared plasmid into E. coli BL21(DE3) to express the protein.

#### Test

##### When the OD of the culture reached 0.4, the culture was induced under the following one of two conditions:

* Incubation with 1 mM IPTG at 37°C for 1.5 hours
* Incubation with 1 mM IPTG at 16°C overnight (o/n)

![](https://static.igem.wiki/teams/5910/eng/eng-fig7.avif)

##### *Figure 7. SDS-PAGE analysis of GST-CARDcasp9 expression Cycle1*

#### Learn

##### Since we could not obtain the expected amount of recombinant protein under multiple culture conditions, we suspected there might be a problem with the plasmid design. Upon investigation, we found that although the amino acid sequence of the GST tag was correct, its nucleotide sequence was different from the original pGEX-6P-1 vector due to codon optimization. We learned that the nucleotide sequence itself, not just the amino acid sequence of the GST portion, can have a critical impact on protein expression.

Cycle2

#### Design

##### Instead of using the construct created with pET21(+), we decided to express GST-CARDcasp9 using the original pGEX-6P-1 vector, which contains the native nucleotide sequence for the GST tag. Our hypothesis was that using the native GST coding sequence would improve protein expression efficiency.

#### Build

##### We excised the CARDcasp9 fragment from the plasmid generated in Cycle 1 using restriction enzyme digestion. This fragment was then ligated into a pGEX-6P-1 vector that had been digested with the same enzymes, resulting in a new plasmid construct. We transformed this plasmid into E. coli BL21(DE3) and proceeded with protein expression.

#### Test

##### The culture was induced with 1 mM IPTG at 37°C for 1.5 hours. Recombinant protein expression was assessed via SDS-PAGE.

![](https://static.igem.wiki/teams/5910/eng/result-figure-11-gst-card-2025-10-09-13-12-59.avif)

##### *Figure 8. SDS-PAGE analysis of GST-CARDcasp9 expression Cycle2*

#### Learn

##### By switching to the pGEX-6P-1 vector with the native GST sequence, we were able to purify a significantly greater amount of recombinant protein. This result highlighted that even when both the amino acid sequence and the promoter remain unchanged, variations in the nucleotide sequence of the open reading frame (ORF)—particularly within regions encoding affinity tags—can dramatically influence the expression yield of recombinant proteins in E. coli.

Cycle3

#### Design

##### Building upon our successful preparation of GST-CARDcasp9 immobilized on Glutathione Sepharose beads, we next designed a pull-down assay to test for interaction with RIG-I-CARDapaf1. To facilitate detection, we inserted a FLAG tag into the RIG-I-CARDapaf1 construct. As a positive control, we planned to test FLAG-tagged CARDapaf1 alone, which was expected to interact with CARDcasp9 based on previous literature.

#### Build

##### We constructed a plasmid encoding FLAG-CARDapaf1 and expressed it in E. coli BL21(DE3). The recombinant protein was purified and its expression confirmed by SDS-PAGE.

#### Test

##### Pull-down assays were performed using Glutathione Sepharose beads bound to GST-CARDcasp9. Proteins that co-precipitated were detected via Western blotting using an anti-FLAG antibody. However, no interaction was observed: neither RIG-I-CARDapaf1 nor FLAG-CARDapaf1 was pulled down.

![](https://static.igem.wiki/teams/5910/eng/eng-fig9.avif)

##### *Figure 9. CARDapaf1-CARDcasp9 interaction assay.*

##### Western blot analysis shows that neither RIG-I-CARDapaf1 nor FLAG-CARDapaf1 was co-precipitated by GST-CARDcasp9.

#### Learn

##### Although a direct in vitro interaction between CARDapaf1 and CARDcasp9 has been previously reported by Tamura et al. (2018) [https://www.nature.com/articles/s41598-018-19845-6], we were unable to replicate this result under our experimental conditions. This suggests that the assay conditions—such as buffer composition, protein concentrations, or wash stringency—may not have been optimal. We concluded that further optimization will be necessary to visualize the interaction between these CARD domains.

References

* Tamura, R., Takada, M., Sakaue, M. et al. Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs. Sci Rep 8, 1611 (2018).

  <https://doi.org/10.1038/s41598-018-19845-6>

# Integration of Two Modules

Initial Plan

##### Our aim was to induce apoptosis through the activation of Caspase-9 by achieving two conditions in our engineered constructs, RIG-I-CARDapaf1 and PKR-CARDapaf1: Binding of double-stranded RNA (dsRNA) via their respective dsRNA-binding domains and Interaction between the CARD domain of Apaf1 and the CARD domain of Caspase-9. To validate that both conditions are met within living cells, we planned to create human or chicken cells stably or transiently expressing these constructs. Upon transfection with dsRNA, we would assess whether apoptosis was successfully induced, thereby verifying the functionality of the integrated module in a cellular context.

Cycle1

#### Design

##### We decided to construct plasmids for transfection into HEK293 and DF-1 cells in order to express our target proteins. Since COCCO is designed to induce apoptosis via oligomerization, we were concerned that overexpression might lead to unintended oligomer formation and premature apoptosis. Therefore, we considered promoter strength optimization to be a critical step.

#### Build

##### Using the CMV promoter, we designed plasmids expressing the following genes:

* RIG-I-CARDapaf1
* PKR-CARDapaf1
* PKR-ΔCaspase9
* non CARD RIG-I
* CARDapaf1
* PKR
* ΔCaspase9
* Empty vector (control)

#### Test

##### We co-transfected these plasmids with pCMV-DsRed into both HEK293 and DF-1 cells. Expression was verified via DsRed fluorescence, and we monitored cells under a microscope to check for signs of apoptosis potentially caused by unintended COCCO activity.

![](https://static.igem.wiki/teams/5910/eng/eng-fig10-1.avif)

![](https://static.igem.wiki/teams/5910/eng/eng-fig10-2.avif)

![](https://static.igem.wiki/teams/5910/eng/eng-fig10-3.avif)

![](https://static.igem.wiki/teams/5910/eng/eng-fig10-4.avif)

##### *Figure 10. Microscopy images of transfected cells.*

##### *The top left image expresses HEK293 expressing RIG-I-CARDapaf1, and the top right one expresses HEK293 expressing DsRed. Also, the bottom left one expresses DF-1 expressing RIG-I-CARDapaf1, and the right bottom one expresses DF-1 expressing DsRed*

#### Learn

##### The successful expression of DsRed confirmed that the transfection and promoter strength were adequate. Moreover, the absence of spontaneous apoptosis in cells expressing COCCO indicated that oligomerization was not occurring inappropriately. From this cycle, we concluded that the CMV promoter provides a suitable expression level for our experiments in both HEK293 and DF-1 cells.

Cycle2

#### Design

##### To integrate the apoptosis-inducing module in mammalian cells, efficient transfection of plasmid DNA is essential. Since our experiments involved two different cell lines—HEK293 (human) and DF-1 (avian)—we recognized the importance of selecting an appropriate transfection method tailored to each. We consulted Dr. Shinnosuke Honda regarding transfection methods suitable for HEK293 and DF-1 cells. Based on his recommendation and prior experience, we chose to use Lipofectamine™ 2000 Reagent for plasmid transfection.

#### Build

##### Following the official Lipofectamine™ 2000 protocol [¹], we transfected our previously constructed plasmids into HEK293 and DF-1 cells. In parallel, we co-transfected pCMV-DsRed to assess transfection efficiency via fluorescence.

#### Test

##### To evaluate the transfection efficiency, we observed DsRed fluorescence in HEK293 and DF-1 cells under a fluorescence microscope.

![](https://static.igem.wiki/teams/5910/eng/eng-fig11-1.avif)

![](https://static.igem.wiki/teams/5910/eng/eng-fig11-2.avif)

##### *Figure 11. Fluorescence microscopy of cells transfected with pCMV-DsRed*

##### *The left image expresses HEK293 expressing DsRed, and the right one expresses DF-1 expressing DsRed.*

#### Learn

##### From the fluorescence images, we observed strong DsRed expression in HEK293 cells, indicating successful transfection and protein expression. However, DF-1 cells exhibited weak or minimal fluorescence. This result suggests that, although plasmid transfection using Lipofectamine™ 2000 is possible in DF-1 cells, it does not lead to sufficient protein expression in this cell line. Therefore, alternative transfection reagents or methods may be necessary for effective expression in DF-1 cells.

Cycle3

#### Design

##### Since the results from Cycle 2 indicated low transfection efficiency in DF-1 cells using Lipofectamine™ 2000, we sought to optimize the protocol specifically for this cell line. Enhancing plasmid delivery efficiency was critical for reliably evaluating downstream apoptosis assays. Based on the observations from Cycle 2, we decided to explore an alternative transfection reagent to improve protein expression in DF-1 cells. Specifically, we compared the transfection efficiencies of Lipofectamine™ 2000 Reagent and FuGENE® 6 Reagent using pCMV-DsRed as a reporter.

#### Build

##### We transfected DF-1 cells with pCMV-DsRed using both Lipofectamine™ 2000 and FuGENE® 6, following each manufacturer's recommended protocol [¹][²].

#### Test

##### To assess transfection efficiency, we observed the fluorescence intensity of DsRed under a fluorescence microscope in DF-1 cells transfected with each reagent.

![](https://static.igem.wiki/teams/5910/eng/eng-fig12-1.avif)

![](https://static.igem.wiki/teams/5910/eng/eng-fig12-2.avif)

##### *Figure 12. Comparison of transfection efficiency between Lipofectamine2000 reagent and FuGENE6 reagent*

##### *The left image expresses DF-1 transfected with DsRed using Lipofectamine, and the right one expresses DF-1 transfected with DsRed using FuGENE.*

#### Learn

##### The fluorescence images showed markedly stronger DsRed expression in DF-1 cells transfected with FuGENE® 6 compared to Lipofectamine™ 2000, indicating improved transfection efficiency. Based on these results, we concluded that FuGENE® 6 is a more suitable transfection reagent for DF-1 cells and will use it in future experiments involving this cell line.

References

* [1] Labmart Ghana. Invitrogen™ Lipofectamine™ 2000 Transfection Reagent.

  [https://www.labmartgh.com/shop/invitrogen-tm-lipofectamine-tm-2000-transfection-reagent-31131 (accessed July 23, 2025)](https://www.labmartgh.com/shop/invitrogen-tm-lipofectamine-tm-2000-transfection-reagent-31131)
* [2] Promega Japan. FuGENE® 6 Transfection Reagent.

  [https://www.promega.jp/products/luciferase-assays/transfection-reagents/fugene-6-transfection-reagent/?catNum=E2691#protocols(accessed August 12, 2025)](https://www.promega.jp/products/luciferase-assays/transfection-reagents/fugene-6-transfection-reagent/?catNum=E2691#protocols )

# Dry Lab Cycle

# Overview

##### To utilize modeling in our project, we performed a viral transmission simulation, which cannot be observed through experiments, and a protein simulation that could also integrate evaluation by the Wet Lab.

# Viral Transmission Modeling

Cycle1

#### Design

##### To prove the effectiveness of COCCO, we divided the viral transmission into three levels: intracellular, intercellular, and inter-individual. Therefore, we set the objective of our initial viral transmission modeling to prove the following points using a mathematical model.

* Within a cell, COCCO causes the host cell to die faster than the virus can proliferate.
* COCCO suppresses viral spread among a cell population, preventing the virus from proliferating to the extent that it can be transmitted to other individuals.
* The spread of viral infection is suppressed within a population of chickens equipped with COCCO.

#### Build

##### At the intracellular level, we analyzed the apoptosis reaction network and simulate that the induction of apoptosis is sufficiently rapid. At the intercellular level, we simulated that increasing the cell death rate suppresses viral proliferation. At the inter-individual level, we simulated that the spread of infection is suppressed because even if infected, individuals transition from an eclipse phase to a susceptible state without becoming infectious. Furthermore, we connected the simulations of these three levels to simulate that the spread of viral infection is suppressed within a chicken coop of COCCO-equipped chickens.

#### Test

##### After reviewing several databases related to metabolic and signaling pathways, we found a lack of detailed information on the apoptosis pathway in chickens, which limited the reliability of intracellular-level modeling.

#### Learn

##### We learned that the chicken apoptosis pathway is not well understood. Therefore, we concluded that the intracellular dynamics modeling could not be sufficiently reliable.

Cycle2

#### Design

##### We decided to exclude the intracellular dynamics modeling and perform a two-level viral transmission simulation for the intercellular and inter-individual levels.

#### Build

##### We decided to use the most widely used TIV model and SIR model to describe the intercellular and inter-individual viral transmission, respectively. We also determined how to proceed with Human Practices to receive advice on necessary assumptions and other points of caution when handling these models.

#### Test

##### In Human Practices (HP link), it was pointed out that the inter-individual viral transmission modeling would not be a reliable simulation unless we used data obtained from our own observations.

#### Learn

##### We realized that we could make almost no claims from the inter-individual viral transmission simulation, as we cannot collect observational data on the spread of viral infection in COCCO-equipped chickens or chicken coops.

Cycle3

#### Design

##### We decided to exclude the inter-individual dynamics modeling and perform a viral transmission simulation at the intercellular level.

#### Build

##### We believed that the time elapsed since a virus entered a cell was important for the simulation of COCCO. Therefore, we introduced the quantity of time known as "infection age" to represent this elapsed time and decided to use an age-structured TIV model[1].

#### Test

##### In Human Practices (HP link), it was pointed out that we should construct a minimal model for our claims and that we should simulate how the infection dynamics change when the apoptosis intensity is varied.

#### Learn

##### We considered the age-structured TIV model to be a minimal model for our purposes and thought that a meaningful modeling approach would be to evaluate the strength of the effect of a decreased death rate on the value that determines the behavior of the infection dynamics.

# Protein Optimization

Cycle1

#### Design

##### To prevent the fusion protein, in which the CARDs of RIG-I were replaced with the CARD of APAF1, from always remaining in an active state that can transmit signals, we decided to predict the optimal amino acid mutations to achieve the ideal function of COCCO through in silico protein simulation.

#### Build

##### We investigated available software and constructed a workflow EVOLVE (version 1) to search for and select the optimal mutant amino acid sequence in the following five steps.

* Mutable Site Selection
* Mutation Introduction
* Structure Prediction
* Visual Inspection
* Docking & Stability Analysis

#### Test

##### We executed this EVOLVE (version 1) and selected five sequences each for the mutated Helicase2i domain and APAF1 CARD domain. The results can be found on the Model page under Protein Optimization.

#### Learn

##### We learned that the workflow allows for the in silico optimization of the amino acid sequences of protein domains to have the functions required for COCCO.

Cycle2

#### Design

##### Based on advice from Human Practices, we learned about the usefulness of a software called ProteinMPNN[2], which predicts amino acid sequences based on 3D structures, and decided to use it.

#### Build

##### We devised EVOLVE (version 2) that uses ProteinMPNN as the software in Step 2: Mutation Introduction.

#### Test

##### We executed this workflow and generated two amino acid sequences with mutations introduced into the Helicase2i domain. The results can be found on the Model page under Protein Optimization.

#### Learn

##### We found that EVOLVE (version 2) could similarly be used to design protein domains with the optimized amino acids required for COCCO.

References

[1]Nelson, P. W., Gilchrist, M. A., Coombs, D., Hyman, J. M., & Perelson, A. S. (2004). An Age-Structured Model of HIV Infection that Allows for Variations in the Production Rate of Viral Particles and the Death Rate of Productively Infected Cells. Mathematical Biosciences & Engineering, 1(2), 267–288.

<https://doi.org/10.3934/mbe.2004.1.267>

[2]Dauparas, J., Anishchenko, I., Bennett, N., Bai, H., Ragotte, R. J., Milles, L. F., Wicky, B. I. M., Courbet, A., De Haas, R. J., Bethel, N., Leung, P. J. Y., Huddy, T. F., Pellock, S., Tischer, D., Chan, F., Koepnick, B., Nguyen, H., Kang, A., Sankaran, B., . . . Baker, D. (2022). Robust deep learning–based protein sequence design using ProteinMPNN. Science, 378(6615), 49–56.

<https://doi.org/10.1126/science.add2187>

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Plasmid Construction

[1. Plasmid-design](#plasmid-design)
[2. Procurement of DNA](#procurement-DNA)
[3. PCR](#PCR)
[4. Gel Preparation](#gel-preparation)
[5. Gel Electrophoresis](#gel-electrophoresis)
[6. DpnI Treatment](#dpnI-treatment)
[7. DNA Purification](#DNA-purification)
[8. DNA Concentration Measurement](#DNA-concentration)
[9. DNA Assembly](#DNA-assembly)
[10. Transformation into E.coli](#transformation)
[11. Direct Colony PCR](#colony-PCR)
[12. Inoculation](#inoculation)
[13. Miniprep](#miniprep)
[14. Sequencing](#sequencing)

Medium Composition

[1. LB liquid medium](#LB-liquid)
[2. LB Agar Plate Preparation](#LB-agar)

Pull-Down Assay

[Pull-Down Assay](#Pull-downAssay)

Ligation

[Ligation](#Ligation)

SDS-PAGE

[SDS-PAGE](#SDS-PAGE)

Cell-related experiments

[1. Thawing frozen cells](#thawing-frozen-cells)
[2. Cell passaging](#cell-passaging)
[3. Transfection Using Lipofectamine 2000](#transfection-lipofectamine)
[4. Transfection Using FuGENE 6](#transfection-fugene)
[5. Creation of Stable Cell Lines](#stable-cell-lines)
[6. Cellular protein harvest](#protein-harvest)
[7. Western blot](#western-blot)

Virus Infection Experiments

[1. Collagen coating](#collagen-coating)
[2. Cell Preparation 1](#cell-preparation-1)
[3. Cell Preparation 2](#cell-preparation-2)
[4. Viral Infection 1](#viral-infection-1)
[5. Viral Infection 2](#viral-infection-2)
[6. MDCK Cell Preparation for Titration](#mdck-cell-preparation)
[7. MDCK Viral Infection (Titration)](#mdck-viral-infection)
[8. FACS Sample Preparation](#facs-sample-preparation)
[9. Fluorescence Observation](#fluorescene-obser)

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## Plasmid Construction

**1. Plasmid-design**

#### plasmid-design

##### We conducted the design of plasmids involved in this project exclusively using Benchling, a platform for molecular biology research and design.

**2. Procurement of DNA**

#### procurement of DNA

##### For this project, we acquired synthetic DNA as follows: All oligoDNA was purchased from Eurofins Scientific. Other synthetic gene fragments were sourced from gBlocks by Integrated Gene Technologies and Gene Fragments by TWIST BIOSCIENCE.

**3. PCR**

#### PCR

##### The following procedure was used to amplify DNA.

1. The sample was prepared as follows:

| Component | Amount(μL) |
| --- | --- |
| Template (diluted to 0.1~1 ng/μL) | 1 |
| Primers (10 uM each) | 0.4 |
| 2x KOD One Blue | 10 |
| Milli-Q water | 8.6 |
| Total | 20 |

##### KOD ONE Blue was provided by Toyobo.

2. PCR was performed with the following thermal cycling conditions:

| Step | Temperature (°C) | Time(s) | Repetitions |
| --- | --- | --- | --- |
| Denaturation | 98 | 10 | 29 |
| Annealing | 55 | 5 | 29 |
| Extention | 68 | ≤1 kb: 1 1~10 kb: 5 / kb | 29 |
| Hold | 4 | pause | -- |

**4. Gel Preparation**

#### Gel Preparation

##### Gels were created for electrophoresis as follows:

1. Agarose was dissolved in 1x TAE buffer at a rate of 1% w/v and the mixture was heated until agarose dissolved.
2. The mixture was poured into a tray with a comb.
3. EtBr was added at a 1:10 ratio to the gel, and gently mixed to ensure even distribution.
4. The gel was allowed to solidify at room temperature.

##### 1x TAE buffer had 40 mM tris-acetate, and 1 mM EDTA. 10 mg/mL EtBr was purchased from Takara Bio Inc.

**5. Gel Electrophoresis**

#### Gel Electrophoresis

##### This procedure was conducted to assess the length of DNA fragments which is amplified through techniques such as PCR.

1. The gel was placed in the electrophoresis chamber without removing it from the tray.
2. 1x TAE buffer was poured into the chamber until the gel was fully immersed.
3. An appropriate amount of size marker was added to the first lane.
4. The samples (3 μL each) were sequentially loaded in the lanes, starting from the second lane.
5. The chamber was turned on and electrophoresis was run at 100 V for 20 minutes.
6. After, the gel was carefully removed from the tray and transferred to another large, transparent tray.
7. The bands were visualized using a CCD camera.

##### Two kinds of size markers were used: λ styI, λ phage DNA fragments cleaved with restriction enzyme EcoT14I, and a mixture of TOYOBO's 100bp ladder and 6x loading dye. 10 μL of the former and 6 μL of the latter were used. The CCD camera was part of the ChemiDoc XRS+ System purchased from Bio-Rad Laboratories.

**6. Dpnl Treatment**

#### Dpnl Treatment

##### DpnI treatment was performed to cleave plasmids propagated in E. coli when they were used as PCR templates. This prevents any residual E. coli-derived plasmid from being carried over.

1. 1 μL of DpnI was added to 20 μL of the PCR product.
2. The mixture was incubated at 37°C for a minimum of 1 hour.

##### DpnI was purchased from Takara Bio Inc.

**7. DNA Purification**

#### DNA Purification

##### DNA purification was conducted to eliminate excess enzymes and residual substances after PCR.

1. 20 μl of the PCR product was mixed with 100 μl Buffer PB in a 1.5 mL tube.
2. This mixture was transferred to a silica membrane column and centrifuged at 14,000 rpm for 1 minute.
3. The supernatant was discarded.
4. 700 μl of Buffer PE was added to the column and centrifuged at 14,000 rpm for 1 minute at room temperature.
5. The supernatant was discarded.
6. The upper portion of the column was attached to a new 1.5 mL tube.
7. DNA was eluted with 10 μl of Milli-Q water.
8. The mixture was left for 1 minute and then centrifuged at 14,000 rpm for 1 minute.
9. Purified DNA was obtained in the 1.5 mL tube.

##### Silica membrane columns were purchased from Aji Bio-Pharma Oligos. Buffer PB and PE were purchased from QIAGEN.

**8. DNA Concentration Measurement**

#### DNA Concentration Measurement

##### To measure the concentration and purity of purified DNA, we used the NanoDrop One spectrophotometer from Thermo Fisher Scientific.

1. The instrument was turned on, and the arm was raised.
2. The platform was wiped with a Kimwipe which is wet with Milli-Q water, and dried by dabbing with a dry Kimwipe.
3. A blank measurement was taken using 1 μL of the same buffer used for the DNA solution.
4. The concentration and purity of the DNA were measured with 1 μL of the DNA solution.
5. After every measurement, the platform was again wiped with a Kimwipe wet with Milli-Q water and dried by dabbing.
6. After all measurements were taken, the arm was lowered, and the instrument was turned off.

**9. DNA Assembly**

#### DNA Assembly

##### We utilized the XE cocktail method (Liu et al., 2023), a low-cost PCR enzyme developed by former members of our team, to combine DNA fragments [1].

1. The thermal cycler was preheated to 37°C.
2. The samples were prepared as follows:

| Component | μL |
| --- | --- |
| Inserts (diluted to 10~30 ng/μL) | 1 each |
| Vector | 1 |
| 2x XE cocktail | total volume of other components |

3. The cocktail was immediately transferred to the thermal cycler and the following program was run.

| Step | Temperature (°C) | Time(min) |
| --- | --- | --- |
| 1 | 37 | pause |
| 2 | 37 | 5 |
| 3 | 65 | 5 |
| 4 | 4 | pause |

4. [1]Liu, A.Y., Koga, H., Goya, C., Kitabatake, M. (2023). Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors. Genes to Cells, 28(8), 553-562. https://doi.org/10.1111/gtc.13034

**10. Transformation into E.coil**

#### Transformation into E.coil

##### We introduced the plasmid into E. coli following this protocol.

1. Up to 2 μl of the assembly product or plasmid was added to 10 μl of competent cells.
2. The mixture was left on ice for 20 minutes.
3. The mixture was placed in a heat block at 42°C for 45 seconds.
4. The mixture was left on ice for 1 minute.
5. 100 μl of SOC medium was added.
6. The mixture was incubated at 37°C for 1 hour in the case of an assembly product, and 45 minutes for the plasmid.
7. The liquid was spread onto a plate following aseptic techniques.
8. The plates were labeled and incubated at 37°C overnight.

Three strains of competent cells were used in this experiment: DH5α, BL21, and BW25113. DH5α was purchased from TOYOBO, and BL21 (DE3) from COSMO BIO CO., LTD. The BW25113 strains were prepared in-house.

**11. Direct Colony PCR**

#### Direct Colony PCR

##### We performed Direct Colony PCR to confirm the presence of the desired plasmid within the colonies grown on an agar plate.

1. The samples were prepared as follows:

| Component | Amount(μL) |
| --- | --- |
| Primers (diluted to 10 mM) | 0.3 |
| 2X KOD ONE Blue | 5 |
| Milli-Q water | To 10 |
| Total | 10 |

2. A single, relatively isolated colony was picked using a pipette tip, and the tip was dipped into the liquid which is prepared as the above in a PCR tube.
3. The DNA was amplified using the following thermal cycling conditions:

| Step | Temperature (°C) | Time(s) | Repetitions |
| --- | --- | --- | --- |
| Pre-heating | 98 | 30 | 1 |
| Denaturation | 98 | 10 | 29 |
| Annealing | 55 | 5 | 29 |
| Extension | 68 | ≤1 kb: 1 1~10 kb: 5 / kb | 29 |
| Hold | 4 | pause | -- |

**12. Inoculation**

#### Inocuration

##### To cultivate a significant quantity of E. coli that is believed to have incorporated the correct plasmid from direct colony PCR, we inoculated them in test tubes with LB medium.

1. 3 mL of LB medium with the appropriate antibiotic was prepared in a test tube.
2. A single colony was picked using a pipette tip and suspended in the prepared medium.
3. The test tube was incubated at 37°C and agitated 180 rpm for at least 16 hours in a shaker.

**13. Miniprep**

#### Miniprep

##### With this method, we extracted the desired plasmid from E. coli cells and purified it. For this procedure, we used the Promega Wizard Plus SV Minipreps DNA purification System.

1. 1.5 mL of the 3 mL culture from the test tube was decanted into a 1.5 mL tube.
2. The 1.5 mL tube was centrifuged at 13,000 rpm for 1 minute, and the supernatant was discarded.
3. Steps 1 and 2 were repeated.
4. The 1.5 mL tube was centrifuged at 13,000 rpm for 1 minute.
5. The supernatant was removed with a pipette.
6. The precipitated cells were fully resuspended with 250 μL of CRA, vortexing, and pipetting.
7. 250 μL of CLA was added, and the solution was mixed with gentle inversion.
8. Once the solution became clear, 10 μL of Alkaline protease was added, and the solution was mixed with inversion. The solution was left for 5 minutes.
9. 350 μL of NSB was added and mixed well by vortexing.
10. The mixture was centrifuged at 13,000 rpm for 10 minutes.
11. An SV minicolumn was placed over a collection tube, and the supernatant was transferred into it.
12. The minicolumn and tube were centrifuged at 13,000 rpm for 1 minute.
13. The supernatant in the collection tube was discarded, and 750 μL of Column Wash Water was added to the tube.
14. The mixture was centrifuged at 13,000 rpm for 10 minutes.
15. The supernatant in the collection tube was discarded, and 250 μL of Column Wash Water was added to the tube.
16. The mixture was centrifuged at 13,000 rpm for 1 minute.
17. The supernatant in the collection tube was discarded, and the collection tube and column were again centrifuged at 13,000 rpm for 2 minutes.
18. The SV minicolumn was placed on the top of a 1.5 mL tube, and DNA was eluted with 50 μL of Milli-Q water.
19. The mixture was centrifuged at 13,000 rpm for 1 minute.
20. The DNA was obtained from the 1.5 mL tube.

**14. Sequencing**

#### Sequencing

##### We conducted sequence analysis using Sanger sequencing provided by Azenta Life Science. All functional regions of the plasmid used in this study were subjected to sequence analysis to verify their sequences.

## Medium Composition

**1. LB liquid medium**

#### LB liquid medium

##### Luria broth medium was used as the medium for the culture of E. coli.

1. The following compounds were mixed in a large glass jar.

| Component | Amount |
| --- | --- |
| Tryptone | 10g |
| Yeast Extract | 5g |
| NaCl | 5g |
| RO Water | 1L |

2. This mixture was autoclaved at 121°C for 15 minutes to sterilize.
3. When it cooled to around 60°C, the appropriate antibiotics were added.

**2. LB Agar Plate Preparation**

#### LB Agar Plate Preparation

##### We prepared LB agar medium to create plates for culturing E. coli.

1. A mixture was prepared in a large glass jar as follows:

| Component | Amount |
| --- | --- |
| Tryptone | 10g |
| Yeast Extract | 5g |
| NaCl | 5g |
| Agar | 12g |
| RO Water | 1L |

2. The mixture was autoclaved at 121°C for 15 minutes to sterilize.
3. When the mixture cooled to around 60°C, it was mixed thoroughly and the appropriate antibiotics were added to it.
4. ~20 mL of the agar medium was poured into each plate and allowed to solidify.

## Pull-Down Assay

**Pull-Down Assay**

#### Pull-Down Assay

1. Add 1 ml of Ni-NTA beads to a 50 ml tube and wash 3 times with Wash Buffer.
2. Adjust the washed beads to 5 ml, then aliquot 250 μl into 1.5 ml tubes.
3. Centrifuge at 13,000 rpm for 1 minute and remove the supernatant.
4. Add the Target DNA and Standard DNA.
5. Add His-tagged protein and adjust to 1 ml with Reaction Buffer.
6. Take 20 μl of the input sample and store it.
7. Incubate at 4°C for 30 minutes.
8. Centrifuge at 13,000 rpm for 1 minute.
9. Remove the supernatant and wash the beads 3 times.
10. Add 100 μl of Milli-Q water to both the input sample and beads, then boil at 95°C for 5 minutes.
11. Centrifuge at 13,000 rpm for 10 minutes.
12. Store the supernatant.

## Ligation

**Ligation**

#### Ligation

| Component | volume(μL) |
| --- | --- |
| Ligation high | 2.5 |
| Vector | 1 |
| Insert | 1.5 |
| total | 5 |

## SDS-PAGE

**SDS-PAGE**

#### SDS-PAGE

| Component | volume(ml) |
| --- | --- |
| 40%Acrylamide | 3.125 |
| 1M Tris pH 8.8 | 3.75 |
| 10% SDS | 0.1 |
| DW | 3.025 |
| total | 10 |

##### Add 100 μL of 10% APS and 10 μL of TEMED, and mix well.

##### Preparation of the Stacking Gel

| Component | volume(ml) |
| --- | --- |
| 40%Acrylamide | 1 |
| 1M Tris pH 8.8 | 1.25 |
| 10% SDS | 0.1 |
| DW | 7.65 |
| total | 10 |

##### Add 100 μL of 10% APS and 10 μL of TEMED, and mix well.

## Cell-related experiments

**1. Thawing frozen cells**

#### Thawing frozen cells

##### Thaw cells that have been frozen at -80°C. Work under disinfect conditions to prevent cell contamination.

1. Add 5 mL of Dulbecco's Modified Eagle Medium (DMEM(-/-)) to the tube and warm it at 37°C.
2. Remove the cells from -80°C, thaw them at 37°C, and then sterilize them with ethanol.
3. Suspend the cells in tube 1 and centrifuge at 800 rpm for 5 minutes at room temperature.
4. Wash once with DMEM (-/-)
5. The supernatant was aspirated and warmed to 37°C. Add 100 μl of complete DMEM medium (hereinafter referred to as DMEM) supplemented with 10% FBS, 1% P/S, and 1% L-Glutamine and suspended.

##### Composition of complete DMEM supplemented with 10% FBS, 1% P/S, 1% L-Glutamine

| Reagent | Volume [ml] |
| --- | --- |
| DMEM(-/-) | 352 |
| Fetal Bovine Serum (FBS) | 40 |
| Penicillin/Streptomycin (P/S) | 4 |
| L-Glutamine | 4 |

6. Seed the cells on a new plate, gently mix to evenly distribute them, and place in an incubator (HEK293: 37℃, DF-1: 38.5℃).

**2. Cell passaging**

#### Cell passaging

##### To maintain healthy cells and promote their proliferation, subculture the cells at the appropriate time when they reach confluence. Work under sterile conditions to prevent cell contamination.

1. Warm DMEM and Phosphate Buffered Saline (PBS) at 37°C and warm Trypsin/EDTA at room temperature.
2. Remove the cells from the incubator and aspirate the old medium from the plate or flask.
3. Wash with PBS
4. Add trypsin/EDTA and incubate until cells detach (HEK293: 37℃, DF-1: 38.5℃).
5. Add DMEM to neutralize the trypsin and collect the cells in a tube.
6. (Optional) Take a small amount of the cell suspension from step 5 and count the number of cells.
7. Centrifuge HEK293 at 900 rpm for 5 minutes, and DF-1 at 1200 rpm for 5 minutes.
8. Aspirate the supernatant and add DMEM to suspend the cells.
9. Add the appropriate amount of DMEM and cells to a new plate or flask, mix gently to evenly distribute, and place in an incubator (HEK293: 37°C, DF-1: 38.5°C).

**3. Transfection Using Lipofectamine 2000 Reagent**

#### Transfection Using Lipofectamine 2000 Reagent

1. Seed cells into appropriate well plates 24 hours prior to transfection and incubate at 37°C for HEK293 and 38.5°C for DF-1 (cells should reach 70-90% confluence).
2. Dilute Lipofectamine 2000 Reagent in Opti-MEM Medium
3. Dilute DNA in Opti-MEM Medium in a separate tube from step 2.
4. Mix the solutions from step 2 and step 3 in a 1:1 ratio and incubate at room temperature for 5 minutes.
5. Add the DNA-lipid complexes to the cells and return the cells to the incubator.

| Component | 96-well | 24-well | 6-well |
| --- | --- | --- | --- |
| DNA-lipid complex per well | 10 μL | 50 μL | 250 μL |
| Final DNA used per well | 100 ng | 500 ng | 2500 ng |
| Final Lipofectamine® 2000 Reagent used per well | 0.2-0.5 μL | 1.0-2.5 μL | 5.0-12.5 μL |

**4. Transfection Using FuGENE 6 Transfection Reagent**

#### Transfection Using FuGENE 6 Transfection Reagent

1. Seed cells into appropriate well plates 24 hours prior to transfection and incubate at 37°C for HEK293 and 38.5°C for DF-1 (cells should reach 50-80% confluence).
2. Add Fugene 6 Reagent to Opti-MEM Medium and incubate at room temperature for 5 minutes.
3. Add plasmid DNA and incubate for 15 minutes.
4. Add the DNA-FuGENE complex to the cells and return the cells to the incubator.

| Component | 96-well | 24-well | 6-well |
| --- | --- | --- | --- |
| DNA-FuGENE complex per well | 5 μL | 25 μL | 100 μL |
| Final DNA used per well | 100 ng | 500 ng | 2000 ng |
| Final Lipofectamine® 2000 Reagent used per well | 0.2-0.5 μL | 1.0-2.5 μL | 5.0-12.5 μL |

**5. Creation of Stable Cell Lines**

#### Creation of Stable Cell Lines

##### Cell lines that stably express the target protein are generated.

1. Transfecting cells with DNA
2. Incubate for 1-2 days
3. Remove the medium from the plate and add DMEM containing 400 μg/mL of G418 to the plate containing the cells.
4. Maintain the cells in DMEM containing 400 µg/mL of G418 at least 2 weeks.

**6. Cellular protein harvest**

#### Cellular protein harvest

##### Protein recovery from cells for SDS-PAGE and Western blotting. Protocol for 96-well plates is shown.

1. Wash the cells with 50 ul/well of ice-cold PBS(-).
2. Add 20 ul/well of ice-cold sample buffer, and transfer all the cells to a PCR tube while scraping the bottom of the dish with a pipette.
   2X sample buffer:
   125mM Tris-HCl buffer(pH6.8)
   4% SDS
   40% Glycerol
   0.01% BP
   10% b-mercaptoethanol (add immediately before use)
3. Wash the well with 10 ul of sample buffer, add to the tube from step 2, vortex, and spin down.
4. Place the spun down sample in a thermal cycler and treat at 95°C for 5 minutes.
5. Store protein samples at -80 °C

**7. Western blot**

#### Western blot

##### Gel making

1. Place the gel plate in the gel preparation bag, clamp it on both sides with acrylic plates, secure the spacer with a clip to prevent it from coming off, and then mark with a pen the area that will be 1 cm below the bottom of the comb when it is inserted.
2. Create a running gel. Mix Acrylamide, Tris, and Milli-Q and vortex. Then add APS and TEMED and mix again.

| Component | Volume (10%) | Volume (12.5%) | Volume (15%) |
| --- | --- | --- | --- |
| 30% AA | 3.3 mL | 4.2 mL | 5.0 mL |
| 1M Tris (pH8.8) | 3.75 mL | 3.75 mL | 3.75 mL |
| 10% SDS | 0.1 mL | 0.1 mL | 0.1 mL |
| DW | 2.817 μL | 2.817 μL | 2.817 μL | 2.817 μL |
| Total | About 10.0 mL | About 10.0 mL | About 10.0 mL | About 10.0 mL |

3. Slowly pour the gel through the gap in the gel preparation bag until the liquid level reaches the pen mark, then slowly add 100 ul Water-saturated butanol from above and let it stand for 20 minutes.
4. Aspirate Water-saturated butanol
5. Prepare the stacking gel in a 15 ml tube with the following composition, similar to the running gel:

| Component | Volume |
| --- | --- |
| 30% Acrylamide | 0.4 mL |
| 0.5M Tris (pH6.8) | 0.75 mL |
| Milli-Q | 1.85 mL |
| 10% APS | 20 μL |
| TEMED | 2 μL |
| Total | About 3.0 mL |

6. Place the stacking gel on top of the running gel (approximately 2.4 mL), insert the comb, and let it stand for 30 minutes.
7. Remove the gel from the gel preparation bag while pouring distilled water over it, taking care not to trap bubbles in the gel, and rinse it lightly with distilled water.

##### Electrophoresis

1. Add 2X sample buffer and 1X PBS in a 1:1 ratio and mix (1X sample buffer).
2. Thaw the protein sample on ice and mix the protein with the sample buffer.
3. Remove the comb from the gel plate, remove any gel adhering to the sides, and place the gel plate in the holder with the notched side facing inward.
4. After placing the gel plate in the container, pour 1X running buffer onto the outside of the gel plate by about 5 cm, and tilt the container to remove any bubbles at the bottom of the gel cassette.
5. Pour 1X running buffer into the gel plate, filling it up to the mark on the outside of the container.
6. Apply sample and ladder to wells
   To prevent smiling, add an equal amount of 1X sample buffer to each empty well.
7. Run at CV (constant voltage) 300V for 15 minutes.

##### Blotting

1. Pour methanol into a Tupperware container and soak the PVSF membrane cut into 45 x 88 mm pieces for about 20 seconds.
2. Transfer the membrane to 1X transfer buffer and shake for at least 15 minutes.
3. Place two pieces of filter paper cut to 45 x 88 mm between the sponges, soak them in 1X transfer buffer in a large Tupperware container, and leave them to stand for at least 5 minutes.
4. After electrophoresis is complete, disassemble the gel plate, cut off the stacking gel and the bottom of the gel, place them in a Tupperware container containing 1X transfer buffer, and shake for 5 minutes.
5. On a large tray, place the layers of material onto the cathode core of the transfer device in the following order: sponge, filter paper, gel, membrane, filter paper, sponge, taking care not to trap air bubbles from below, and finally place the anode core of the transfer device on top.
6. Place the transfer device horizontally into the electrophoresis chamber, fix it with a wedge, then turn it vertically and fill it up with a new 1X transfer buffer.
7. Chill on ice and run at CV (constant voltage) 30V for 15 minutes.

##### Blocking and primary antibody

1. Prepare a blocking solution (5% BSA) by dissolving 0.5 g of BSA (for blocking) in 10 mL of PBST.
2. Add 2 mL of blocking solution to 8 mL of PBST to make antibody dilution solution (1% BSA).
3. After the transfer is complete, pour 10 mL of PBST into a Tupperware container, immerse the membrane with the transferred side facing up using tweezers, and shake on a shaker at room temperature for 10 minutes.
4. Pack the membrane in 3 mL of blocking solution using plastic film or a 25 mL tube and rotate at room temperature for 1 hour.
5. Dilute the primary antibody to an appropriate concentration in an appropriate amount of antibody diluent (minimum 3 mL for a 25 mL tube), pack the membrane removed from blocking in the same manner as blocking, and rotate it at 4°C overnight.

##### Secondary antibody and ECL reaction

1. Pour 10 mL of PBST into a Tupperware container, immerse the membrane in it, and shake it at room temperature for 10 minutes. Repeat this process three times.
2. Dilute the secondary antibody in antibody diluent to an appropriate concentration (Cytiva antibodies are 1:10,000), pack the membrane in the same manner as for blocking, and rotate at room temperature for 1 hour.
3. Pour 10 mL of PBST into a Tupperware container, immerse the membrane in it, and shake it at room temperature for 10 minutes. Repeat this process three times.
4. Mix ECL reagent A and B in a 1:1 ratio in an Eppendorf tube.
   \*Required ECL mixture is 50–100 µl/cm²
5. Place the membrane in a plastic container for measuring, add the ECL mixture and shake gently to spread it evenly.
6. Place the membrane in the scanner with the protein-adsorbed side facing down, absorb excess ECL mixture, and then scan.

## Virus Infection Experiments

**1. Collagen Coating**

#### Collagen Coating

1. Prepare collagen solution and add 50 µL/well to 96-well plates.
2. Incubate at room temperature on a shaker for at least 1 hour.

**2. Cell Preparation 1**

#### Cell Preparation 1

##### Use DF-1 cells:

1. Aspirate 10% FBS/DMEM from flasks.
2. Wash twice with PBS.
3. Add trypsin and incubate at 38.5°C for 5 min.
4. Add 2 mL DMEM and resuspend cells by pipetting; transfer to 5 mL tubes.
5. Centrifuge at 1200 rpm for 5 min.
6. Aspirate supernatant, gently resuspend pellet by flicking the tube, add 0.5 mL 10% FBS/DMEM, and pipette to mix.
7. Take 10 µL for automated cell counting. Note: The counting solution contains 5 µL of dye and 5 µL of cell suspension, so divide the displayed cell number by 2.
8. Adjust cell concentration to 5.0 × 105 cells/mL.
9. Remove prepared collagen solution from wells.
10. Seed 0.1 mL/well of the cell suspension into 96-well plates (3 wells per cell type).
11. Incubate at 38.5°C with 5% CO2.

**3. Cell Preparation 2**

#### Cell Preparation 2

##### Use HEK293 cells:

1. Aspirate 10% FBS/DMEM from flasks.
2. Wash twice with PBS.
3. Add 0.5 mL trypsin and incubate at 37°C for 5 min.
4. Add 2 mL DMEM, pipette to resuspend, transfer to a 5 mL tube.
5. Centrifuge at 900 rpm for 5 min.
6. Aspirate supernatant, resuspend pellet in 0.5 mL 10% FBS/DMEM, pipette gently.
7. Take 10 µL for automated counting (divide by 2 due to dye dilution).
8. Adjust to 4.0 × 105 cells/mL.
9. Remove collagen from wells and seed 0.1 mL/well.
10. Incubate at 37°C with 5% CO2.

**4. Viral Infection 1**

#### Viral Infection 1

| Reagents | volume(μl) |
| --- | --- |
| Dk/Hok/8/80(2.4\*108PFU/ml) | 380 |
| BSA/DMEM | 8740 |
| total(1.0\*107PFU/ml) | 9120 |

1. Wash cells once with BSA/DMEM.
2. Dilute virus in BSA/DMEM to achieve 1 × 107 PFU/mL (5.0 × 104 cells/well × 10 MOI / 0.050 mL).
3. Remove BSA/DMEM and add virus solution.
4. Incubate at 38.5°C for 1 hour for viral adsorption.
5. Remove virus solution and wash once with BSA/DMEM.
6. Add BSA/DMEM and incubate at 38.5°C.

**5. Viral Infection 2**

#### Viral Infection 2

| Reagents | volume(µl) |
| --- | --- |
| A/Victoria/361/2011(9.0\*108PFU/ml) | 56 |
| BSA/DMEM | 6244 |
| total(8.0\*106PFU/ml) | 6300 |

1. Wash cells once with BSA/DMEM.
2. Dilute virus in BSA/DMEM to 8 × 106 PFU/mL (4.0 × 104 cells/well × 10 MOI / 0.050 mL).
3. Remove BSA/DMEM, add 50 µL virus solution; mock wells receive 100 µL BSA/DMEM.
4. Incubate at 37°C for 1 hour for adsorption.
5. Remove virus solution, wash once with BSA/DMEM, then add BSA/DMEM and incubate at 37°C.

**6. MDCK Cell Preparation for Titration**

#### MDCK Cell Preparation for Titration

1. Aspirate medium, wash twice with PBS.
2. Cells were sequentially treated with trypsin to achieve complete detachment, with incubations of 2 mL (immediate removal), 2 mL (7 min + 5 min), 1 mL (5 min), and 1 mL (3 min).
3. Add 5% FCS/DMEM, pipette to resuspend cells, and transfer to a tube.
4. Centrifuge at 900 rpm for 5 min.
5. Aspirate supernatant, resuspend in 10 mL 5% FCS/DMEM.
6. Take 10 µL and transfer to a counting chamber for cell counting.
7. Adjust to 2.5 × 105 cells/mL.
8. Seed 100 µL/well into 96-well plates and incubate at 37°C with 5% CO2.

**7. MDCK Viral Infection (Titration)**

#### MDCK Viral Infection (Titration)

##### Use 9 hpi samples.

1. Thaw 100 µL virus.
2. Aspirate MDCK medium, wash twice with BSA/DMEM, incubate at 37°C.
3. Add 100 µL TPCK trypsin to BSA/DMEM 75 mL.
4. Add 135 µL/well to new 96-well plates.
5. Perform 8-step 10-fold serial dilution by adding 15 µL virus per 3 wells per sample, transferring sequentially.
6. Aspirate BSA/DMEM from MDCK cells, add 100 µL diluted virus per well.
7. Incubate at 37°C with 5% CO2.

**8. FACS Sample Preparation**

#### FACS Sample Preparation

##### Collect cells at 3, 6, 9, 12, and 14 hpi for analysis.

| Reagents | volume(µl) |
| --- | --- |
| PBS | 100 |
| Hoechst | 0.1 |
| FVD | 0.1 |

| Reagents | volume(µl) |
| --- | --- |
| Annexin Binding Buffer | 100 |
| Annexin V-FITC | 0.4 |

1. Preparation of staining reagents.
2. Aspirate culture medium and wash cells once with PBS.
3. For pCK007, pCK008, pCK800, and Wild type at 9 and 12 hpi, collect supernatant for viral titration.
4. Add trypsin and resuspend cells in 10% FBS/DMEM.
5. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
6. Add PBS staining solution containing Hoechst and FVD(+), vortex gently, and incubate 15 min at room temperature in the dark.
7. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
8. Add Annexin V solution, vortex gently, incubate 15 min at room temperature in the dark.
9. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
10. Add Annexin Binding Buffer, vortex gently.
11. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
12. Add 1% formaldehyde in Annexin Binding Buffer and fix cells on ice for 10 min in the dark.
13. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
14. Add Annexin Binding Buffer, vortex gently.
15. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
16. Add Annexin Binding Buffer, vortex gently.
17. Store tubes in sample boxes wrapped in aluminum foil at 4°C.

##### **Virus inactivation:**

##### Two days post-collection, add 1% formaldehyde/Annexin Binding Buffer to inactivate virus. Centrifuge at 1900 rpm for 5 min, aspirate, and wash twice with Annexin Binding Buffer.

**9. Fluorescence Observation**

#### Fluorescence Observation

##### Observe MOCK, 6 hpi, 9 hpi, 12 hpi samples.

| Reagents | volume(µl) |
| --- | --- |
| Annexin Binding Buffer | 100 |
| PI | 0.2 |
| Annexin V-FITC | 0.4 |
| Hoechst | 0.1 |

1. Prepare staining solution.
2. Wash cells once with PBS and add 100 µL staining solution per well.
3. Incubate 20 min at room temperature in the dark.
4. Observe under a fluorescence microscope.

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Overview

Biochemical Characterization

[1. RIG-I-CARDapaf1](#evaluation-rigi)
[2.Evaluation of RIG-I-CARDapaf1 Variants](#rigi-cardapaf1)
[Conclusion](#conclusion-biochem)

Experimental verification using cultured cell lines

[1. Apoptosis
induction by dsRNA transfection](#apoptosis-dsrna)
[2.
Infection Experiment](#infection-experiment)
[Conclusion](#conclusion-experimental)

References

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# Overview

##### Our goal was to establish proof of concept for COCCO in chicken cultured cells. To this end, we planned to demonstrate that, upon viral infection, chicken cells expressing COCCO undergo dsRNA-dependent apoptosis.

##### In this study, we examined four COCCO subtypes (see Design page). The objective of the experiments described below was to compare the performance of these four COCCO constructs across multiple assays and thereby define the conditions under which COCCO fulfills the required functions.

##### For the COCCO fusion protein to achieve its function, two elements are required: (1) the dsRNA sensor must bind dsRNA, and (2) the apoptosis inducer must interact with downstream apoptosis-related proteins. We evaluated each of these two functions in a series of in vitro experiments. In parallel, we generated human and chicken cell lines that constitutively express COCCO. We transfected these cells with dsRNA as a pseudo-infection stimulus and assessed the extent to which apoptosis was promoted relative to control cells. We also infected these cells with influenza virus and similarly evaluated the extent to which apoptosis was promoted compared with

##### We assessed these two essential functions through in vitro assays designed to evaluate dsRNA binding and protein–protein interaction capabilities of the COCCO fusion proteins.

##### In parallel, we established human (HEK293) and chicken (DF-1) cell lines stably or transiently expressing each COCCO construct. We then introduced synthetic dsRNA into these cells to mimic viral infection and compared apoptosis induction levels with those in control cells. Furthermore, we infected the same cell lines with influenza virus to assess whether COCCO expression enhanced virus-induced apoptosis compared to controls.

# Biochemical Characterization

## 1.RIG-I-CARDapaf1

![](https://static.igem.wiki/teams/5910/results/img1-c.avif)
![](https://static.igem.wiki/teams/5910/results/img1-d.avif)
![](https://static.igem.wiki/teams/5910/results/img1-a.avif)
![](https://static.igem.wiki/teams/5910/results/img1-b.avif)

*Figure1. Four COCCO fusion constructs*

##### Among the four fusion proteins we designed to induce apoptosis in a dsRNA-dependent manner, PKR-CARDapaf1 has been previously constructed and demonstrated as a proof-of-concept system [[1]](#references). In contrast, RIG-I-CARDapaf1 is a newly designed construct developed in this study. It consists of the RIG-I domain, which recognizes and oligomerizes upon viral dsRNA binding [[2]](#references), and the CARD domain of Apaf1, which recruits Caspase-9 to trigger apoptosis [[3]](#references).

##### Because RIG-I recognizes different dsRNA substrates compared to PKR, it may complement existing systems such as DRACO (which uses PKR) and thus broaden the antiviral spectrum when used in combination. However, RIG-I is also subject to complex autoinhibitory regulation, where interactions between its CARD domain and Hel2i domain dynamically change depending on the presence of dsRNA. This raises the concern that simply swapping the CARD domain with that of Apaf1 may not suffice to ensure signal transduction upon dsRNA binding.

##### For this reason, we focused specifically on validating the functionality of RIG-I-CARDapaf1, and evaluated two critical properties through in vitro assays:

* Binding of the RIG-I domain to dsRNA
* Interaction between CARDapaf1 and the CARD domain of Caspase-9 (CARDcasp9)

(Note: CARDcasp9 refers to the Caspase-9 domain that interacts with Apaf1's CARD [[4]](#references).)

#### dsRNA-RIG-I interaction assay

##### main achievements

* We established an experiment to synthesize dsRNA and examine its binding to RIG-I.
* We purified RIG-I-CARDapaf1 and its mutant proteins.

![](https://static.igem.wiki/teams/5910/results/img02.avif)

*Figure2. Helicase–dsRNA Interaction Diagram*

##### We conducted an experiment to confirm the binding of dsRNA with RIG-I–CARDapaf1.Specifically, we prepared His-tagged recombinant RIG-I–CARDapaf1 and incubated it with dsRNA in buffer. Thereafter, we pulled down RIG-I–CARDapaf1 using nickel beads. The co-precipitated dsRNA was extracted and quantified by qRT-PCR.

**view details**

##### >>Protein Purification

##### We constructed a plasmid that expresses His-tagged RIG-I-CARDapaf1. The figure below shows the domains of RIG-I-CARDapaf1.

![](https://static.igem.wiki/teams/5910/results/img03.avif)

*Figure3. Domain architecture of RIG-I-CARDapaf1*

##### Following transformation of the plasmids into BL21(DE3) and subsequent protein purification, SDS–PAGE analysis revealed the expected bands for the target proteins, as shown in fig4.

![](https://static.igem.wiki/teams/5910/results/img04.avif)

*Figure4. SDS–PAGE analysis of RIG-I–CARDapaf1*

RIG-I–CARDapaf1 appears around 110 kDa.

#####

##### >>dsRNA synthesis

##### Based on prior reports, template DNA for dsRNA synthesis was custom-ordered from IDT. Two complementary templates were designed such that each insert was flanked on both sides by oppositely oriented T7 promoters, enabling in vitro transcription of both strands from a single construct.

![](https://static.igem.wiki/teams/5910/results/img05.avif)

*Figure5. Template DNA for in vitro dsRNA*

##### dsRNA was generated from the template DNA using an in vitro transcription kit. As shown in the figure, a band was obtained at approximately the expected length. From a 20 uL transcription reaction, 36 ug of RNA was recovered.

![](https://static.igem.wiki/teams/5910/results/fig6new.avif)

*Figure6. Gel electrophoresis of dsRNA*

*A band corresponding to 512 bp dsRNA was observed.*

##### Results of the pull-down qRT-PCR

##### Purified RIG-I-CARDapaf1 and mutRIG-I-CARDapaf1 were incubated with dsRNA to observe their binding interactions. After incubation, mixtures were captured using Ni–NTA agarose pull-down. RNA was then extracted from the beads, and the recovered RNA was quantified using qRT-PCR. As a negative control, a sample containing only Ni-NTA agarose and dsRNA was prepared, and the difference was compared.

##### >>pull-down assay

#####

![](https://static.igem.wiki/teams/5910/results/img07.avif)

*Figure7. RT-PCR*

*dsRNA was precipitated using RIG-I–CARDapaf1. For samples labeled ‘mut’, see
the next section for details.*

##### As a result of RT-PCR, it was found that when the negative control prepared as beads only was reacted with dsRNA, the amount of dsRNA that precipitated fell down was the highest. From the above results, in the present experiment a significant precipitation of dsRNA by RIG-I–CARDapaf1 was not detected. Reasons can be considered, for example, that the experimental conditions are not optimized. Since it appears that a certain amount of dsRNA is precipitating even with beads only, the possibility that the washing of the beads was insufficient is high. Each sample was tested three times and error bars were attached, but the variation of the results was very large, and from this as well, our conclusion—that due to problems in experimental technique we are not able to observe the correct results—is supported.

##### Creating cDNA targeting dsRNA and performing qRT-PCR was a challenging task. However, as described above, the quantification itself was successful, and we expect that with optimization of the pull-down, correct conclusions will be obtained in the future.

![](https://static.igem.wiki/teams/5910/results/img08.avif)

*Figure8. Measurement of the standard curve using dsRNA standard samples*

*A dilution series of synthesized dsRNA was prepared, and the standard
curve was measured by real-time PCR.*

#### CARDapaf1-CARDcasp9 interaction assay

![](https://static.igem.wiki/teams/5910/results/img09.avif)

*Figure9. CARD–CARD interaction diagram*

##### We performed an experiment to verify binding between RIG-I-CARDapaf1and CARDcasp9. Specifically, GST-tagged CARDcasp9 was immobilized on beads and incubated with RIG-I-CARDapaf1 for sufficient time to allow interaction. The mixture was then centrifuged to precipitate GST–CARDcasp9. The precipitates were separated by SDS–PAGE, and Western blotting was used to determine whether the RIG-I construct co-precipitated. For this purpose, the RIG-I construct was designed with an inserted Flag tag.

##### main achievements

* Established a method to evaluate the interaction between CARDapaf1 and CARDcasp9.

**view details**

##### >>Protein Purification

##### By following the published precedent we constructed a plasmid encoding GST-tagged CARDcasp9.

![](https://static.igem.wiki/teams/5910/results/img10.avif)

*Figure10. Nucleotide sequence of the GST-CARDcasp9 expression construct*

##### As shown in the figure, SDS–PAGE revealed a band corresponding to the target protein.

![](https://static.igem.wiki/teams/5910/results/img11.avif)

*Figure11. Result of SDS-PAGE*

##### >>pull-down assay

![](https://static.igem.wiki/teams/5910/results/img12.avif)

*Figure12. Result of Western blotting*

*A band corresponding to the target protein appears near 110 kDa. A band
near 20 kDa likely represents a degradation fragment.*

##### Using GST-tagged fusion CARDcasp9 immobilized on glutathione–Sepharose beads, we pulled down RIG-I–CARDapaf1. The pulled-down RIG-I was detected by Western blotting using an anti-Flag antibody. The results of the Western blotting are shown in Figure12.

##### RIG-I–CARDapaf1 showed no co-precipitation. Because CARDapaf1 is originally a construct that has been reported to co-precipitate with GST–CARDcasp9, this was an unexpected result. Two possibilities can be considered as reasons for this. One is the possibility that the recombinant protein of RIG-I–CARDapaf1 is unstable and could not maintain sufficient interaction during the assay. The other is the possibility that because the RIG-I portion is fused unlike in the paper, steric hindrance prevented the CARDapaf1–CARDcasp9 interaction. Indeed, in the input signal, the expected 110 kDa band was faint, and an unexpectedly larger protein band was observed. This suggests the possibility that the folding of the fusion protein was not appropriate or that aggregation was occurring.

##### For non-CARD–RIG-I, precipitation was observed even when CARDcasp9 was not bound to the beads. We consider that this is because this fusion protein has a property of readily precipitating.

##### From the above results, we did not succeed in observing an interaction between the target RIG-I–CARDapaf1 and GST–CARDcasp9. We consider that this is attributable to the instability of the recombinant proteins. In COCCO, because the recombinant proteins are expressed in cells without purification, we expect that such problems can be avoided.

## 2.Evaluation of RIG-I-CARDapaf1 Variants

![](https://static.igem.wiki/teams/5910/results/img13.avif)

*Figure13. Interactions of each CARD with partner proteins*

##### In addition, we undertook improvements to RIG-I–CARDapaf1. In cells that overexpress RIG-I–CARDapaf1, there is a possibility that RIG-I–CARDapaf1 may accidentally oligomerize in a manner independent of dsRNA and cause dsRNA-independent apoptosis.Therefore, we aimed to prevent dsRNA-independent apoptosis by making CARDapaf1 and HEL2i (helicase domain) bind when dsRNA is absent, so that CARDapaf1 would not be exposed. By this means, even if the fusion protein aggregates in the absence of dsRNA, oligomerization between CARDapaf1 molecules will not occur, and the downstream apoptotic pathway will not be activated. In wild-type RIG-I in the absence of dsRNA, binding between the helicase domain and the intrinsic CARD of RIG-I (CARDrig-i) prevents exposure of CARD RIG-I and suppresses downstream activation [[2]](#references). We attempted to reproduce this mechanism.

##### By in *silico* protein engineering, we designed a CARDapaf1 mutant (RIG-I-mutCARDapaf1) in which mutations were introduced so that CARDapaf1, which does not originally bind the RIG-I helicase domain, acquires binding ability to the helicase domain. We also designed a helicase mutant (mutRIG-I-CARDapaf1)in which mutations were introduced into the helicase domain so that it acquires binding ability to CARDapaf1. To confirm whether these mutant proteins had acquired the intended binding capabilities, we verified the following three properties by in vitro experiments.

* Binding between mutRIG-I–CARDapaf1 and dsRNA
* Binding between CARDapaf1of RIG-I–mutCARDapaf1 and CARDcasp9
* Intramolecular binding between CARDapaf1 and the helicase domain within
  mutRIG-I–CARDapaf1

#### dsRNA-murRIG-I interaction assay

##### main achievements

* We designed mutants of RIG-I-CARDapaf1 by modeling
* We purified the designed mutants and assayed its binding with dsRNA.

![](https://static.igem.wiki/teams/5910/results/img14.avif)

*Figure14. Helicase–dsRNA Interaction Diagram*

#####

**view details**

##### >>Protein Purification

![](https://static.igem.wiki/teams/5910/results/img15.avif)

*Figure15. Domain architecture of mutRIG-I–CARDapaf1*

*The mutated amino acids were selected based on the three-dimensional
structure, as described in the Modeling section. We chose residues predicted to be directly involved
in the CARD–HEL2i interaction.*

*These plasmids were transformed into BL21(DE3), and upon protein
purification, SDS–PAGE revealed bands corresponding to the target proteins, as shown in Figure16.*

#####

![](https://static.igem.wiki/teams/5910/results/img16.avif)

*Figure16. SDS–PAGE of mut-RIG-I–CARDapaf1*

##### >>dsRNA preparation

##### The dsRNA generated for the experiments in the preceding section was also used in this experiment.

##### >>Pull-down assay

##### As in the preceding section, we performed a pull-down assay and quantified the amount of dsRNA precipitated with mutRIG-I–CARDapaf1.

![](https://static.igem.wiki/teams/5910/results/img17.avif)

*Figure17. Result of RT-PCR*

##### Using RIG-I–CARDapaf1 or mutRIG-I–CARDapaf1 bearing mutations in HEL2i, we pulled down dsRNA.

##### RT-PCR analysis revealed that the negative control (beads only) recovered the highest amount of dsRNA, indicating some inadequacies, such as insufficient washing. Therefore, we cannot draw conclusions about specific protein-dsRNA binding from this experiment.

##### Interestingly, mutRIG-I–CARDapaf1 with mutations in HEL2i consistently yielded less recovered dsRNA than RIG-I–CARDapaf1 with wild-type RIG-I. This may indicate that introducing mutations into HEL2i, which is the dsRNA-binding domain, reduced dsRNA-binding ability. In that case, introducing mutations into HEL2i would decrease performance as a dsRNA sensor, and thus it may be more appropriate to introduce mutations into CARDapaf1 to enhance the CARDapaf1–HEL2i interaction rather than mutating HEL2i itself.

#### mutCARDapaf1-CARDcasp9 interaction assay

![](https://static.igem.wiki/teams/5910/results/img18.avif)

*Figure18. CARD–CARD Interaction Diagram*

##### We conducted an experiment to confirm that binding occurs between RIG-I-mutCARDapaf1 and CARDcasp9. The RIG-I construct was designed with an inserted FLAG tag for the purpose of detecting co-precipitation. Specifically, GST-tagged CARDcasp9 was bound to beads and mixed with RIG-I-mutCARDapaf1, incubated for sufficient time for their interaction, and then GST-CARDcasp9 was precipitated by centrifugation. Subsequently, the precipitate was separated by SDS-PAGE, and Western blotting was used to determine whether the RIG-I construct had co-precipitated

##### main achievements

* We purified RIG-I–mutCARDapaf1

**view details**

##### >>Protein Purification

##### GST-tagged CARDcasp9 was purified as described above.

![](https://static.igem.wiki/teams/5910/results/img19.avif)

*Figure19. Domain architecture of GST-CARD*

##### Multiple plasmids expressing RIG-I–mutCARDapaf1 (bearing mutations in CARDapaf1) were constructed, transformed into BL21(DE3), and the proteins were purified. As shown in the figure, SDS–PAGE revealed bands corresponding to the target protein.

![](https://static.igem.wiki/teams/5910/results/img19.avif)

*Figure20. Result of SDS-PAGE*

##### >>pull-down assay

![](https://static.igem.wiki/teams/5910/results/img21.avif)

*Figure21. Result of Western blotting*

##### Using GST-tagged CARDcasp9 immobilized on glutathione–Sepharose beads, we pulled down RIG-I–mutCARDapaf1. The pulled-down RIG-I–mutCARDapaf1 was detected by Western blotting using an anti-Flag antibody. The results of the Western blotting are shown in Figure21.

##### mutRIG-I–CARDapaf1 (the band near 110 kDa) included cases that, like ΔCARD RIG-I, precipitated in the tube due to aggregation regardless of the presence of GST–CARDcasp9 (mut2 and mut4), and cases that, like RIG-I–CARDapaf1, showed no precipitation under any condition (mut3, mut5).

##### From the above results, we did not reach the point of observing an interaction between mutRIG-I–CARDapaf1 and GST–CARDcasp9. As discussed for the wild-type protein in the previous section, we consider that this is attributable to the instability of the recombinant proteins. In COCCO, because the recombinant proteins are expressed in cells without purification, we expect that such problems can be avoided.

#### CARD-Helicase interaction assay

![](https://static.igem.wiki/teams/5910/results/img22.avif)

*Figure22. CARD-Helicase interaction diagram*

##### The mutRIG-I-CARDapaf1, which carries mutations in the helicase domain, was designed through modeling with the expectation that the helicase domain would interact with its own CARDapaf1. In this study, these proteins were expressed in *E. coli* and purified as recombinant proteins to experimentally verify whether the intended properties were conferred. Specifically, a GST-tagged portion of the helicase domain of mutRIG-I-CARDapaf1 (hel2i) was generated and bound to glutathione beads. These beads were then used to perform a pull-down assay with Flag-tagged CARDapaf1 (Flag-CARDapaf1) to test for the intended interaction.

##### main achievement

* The GST-tagged hel2i, including the mutants, was purified.
* An experimental method to evaluate the interaction between hel2i and CARD was established.

**view details**

##### >>Protein Purification

##### The hel2i2 fragment was amplified by PCR from the plasmid expressing mutRIG-I-CARDapaf1 and assembled into pGEX-6p-1 to generate a plasmid expressing GST-tagged hel2i2.

![](https://static.igem.wiki/teams/5910/results/img23.avif)

*Figure23. Sequence of GST-hel2i2*

##### Flag-CARDapaf1 purified in the previous section was used. In the case of wild-type RIG-I, the hel2i domain interacts with its own CARD. As a positive control, a plasmid expressing the CARD domain of RIG-I with a Flag tag (Flag-CARDrig-I) was constructed.

![](https://static.igem.wiki/teams/5910/results/img24.avif)

*Figure24. Sequence of Flag-CARDrig-I*

##### These plasmids were transformed into BL21(DE3) for expression, and the recombinant proteins were purified. As shown in the figure, SDS-PAGE revealed bands corresponding to the target proteins.

![](https://static.igem.wiki/teams/5910/results/img25.avif)

*Figure25. SDS-PAGE of GST-hel2i purification*

##### >>pull-down assay

![](https://static.igem.wiki/teams/5910/results/img26.avif)

*Figure26. Result of western blotting using His-Flag-CARDapaf1*

*Flag-CARDapaf1 was observed as a band of approximately 10–17 kDa.*

![](https://static.igem.wiki/teams/5910/results/img27.avif)

*Figure27. Result of western blotting using His-Flag-CARDrigI*

*Flag-CARDrigI was observed as a band of approximately 10–17 kDa.*

##### Flag-CARDapaf1 was pulled down using GST-tagged hel2i immobilized on glutathione sepharose beads. As a positive control, His-Flag-CARDrigI, which is expected to interact with RIG-I hel2i, was also pulled down. The pulled-down proteins were detected by western blotting. The results of the western blotting are shown in Figure26 and 27.

##### His-Flag-CARDrigI bound to both wild-type and mutant hel2i. However, His-Flag-CARDapaf1 did not bind to either wild-type or mutant hel2i.

##### Although the interaction between the hel2i mutants and CARDapaf1 was not observed, the differences in the intensity of His-Flag-CARDrig-i bands precipitated by the hel2i mutants indicate that the mutations designed through modeling were able to modulate hel2i’s binding to CARD.

## Conclusion

##### Through extensive trial and error, we successfully expressed and purified many of the required recombinant proteins. Notably, we succeeded in preparing RIG-I, which has been considered difficult to express and purify in *E. coli*, representing a significant advancement for improving the efficiency of future experiments. We also established protocols for dsRNA purification and pull-down assays. Although optimization of the conditions was not completed in time and the intended binding could not be clearly observed, we were able to demonstrate essential steps for the progression of the project, including the ability to precipitate dsRNA via pull-down and the feasibility of accurate quantification using qRT-PCR targeting the dsRNA region.

##### An exciting approach was also undertaken in which mutants were designed through modeling and their properties were subsequently validated experimentally. While this did not generate the novel protein-protein interactions that were anticipated, we observed that the original protein-protein interactions were successfully reduced as expected. This outcome suggests that, with further refinement of the methods, efficient artificial evolution of new proteins may be achievable.

# Experimental verification using cultured cell lines

## 1. Apoptosis induction by dsRNA transfection

![](https://static.igem.wiki/teams/5910/results/img28.avif)

*Figure28. dsRNA transfection diagram*

##### COCCO is a system that rapidly induces apoptosis in response to viral double-stranded RNA (dsRNA) in cells. In this experiment, we used human and chicken cultured cell lines (HEK293 and DF-1) that constitutively express COCCO to verify whether dsRNA-dependent apoptosis occurs through COCCO activity.

##### We constructed plasmids carrying Composite Parts encoding COCCO fusion proteins and parts for negative control experiments. Then, we established human cultured cells (HEK293) and chicken cultured cells (DF-1) that constitutively express COCCO and each control protein. Finally, we introduced dsRNA synthesized by in vitro transcription into the COCCO-expressing cells and observed whether COCCO induces apoptosis in the presence of dsRNA.

##### For comparison, we conducted an experiment in which the protein reagent DRACO was expressed in *E. coli*, purified according to the literature, and transfected into cell lines to examine its effects [[2]](#references). DRACO is a protein in which a cell-penetrating tag such as TAT is fused to PKR-CARDapaf1. This allows DRACO purified outside the cells to be introduced into the cells instead of being expressed intracellularly.

##### main achievements

* We purified DRACO and performed a replication experiment.
* We investigated whether COCCO induces apoptosis through dsRNA transfection.

**view details**

##### >>Protein Purification

![](https://static.igem.wiki/teams/5910/results/img29.avif)

*Figure29. Domain architecture of PKR-CARDapaf1*

##### Based on the literature, we designed a plasmid expressing DRACO (TAT-PKR-CARDapaf1) as shown in Figure29.

![](https://static.igem.wiki/teams/5910/results/img30.avif)

*Figure30. SDS-PAGE of DRACO purification*

##### The plasmid was transformed into BL21 cells to express DRACO (TAT-PKR-CARDapaf1). The recombinant protein was purified using Ni-NTA sepharose through the His tag. As shown in Figure30, a band was observed by SDS-PAGE.

##### >>Reproduction experiment of DRACO

![](https://static.igem.wiki/teams/5910/results/img31-1.avif)
![](https://static.igem.wiki/teams/5910/results/img31-2.avif)

*Figure31. Result of reproduction experiment*

*In DF1 cells, the fractions of PI-positive cells and Annexin
V-positive cells were measured after transfection with each of the following: DRACO stored at −80 ° + dsRNA, DRACO stored at 4 °C + dsRNA, DRACO only, dsRNA only, and non-treated control. dsRNA is 25 ng/well.

* A, Fraction of PI-positive cells in HEK293 under each condition.
* B, Fraction of Annexin V-positive cells in HEK293 under each condition.
* C, Fraction of PI-positive cells in DF1 under each condition.
* D, Fraction of Annexin V-positive cells in DF1 under each condition.*

##### Using the prepared dsRNA and DRACO, we performed a reproduction experiment of DRACO. Specifically, both DRACO and dsRNA were transfected into HEK293 and DF1 cells using FuGENE 6 Reagent. The former is a human-derived cell line known for its high transfection efficiency, making it suitable for simple characterization of our parts. The latter is a chicken-derived cell line that was chosen because it allows observation of infection and propagation of avian influenza virus.

##### Twenty-four hours after transfection, we attempted to observe DRACO-induced, dsRNA-dependent apoptosis by staining the cells with PI and Annexin V and observing them under a fluorescence microscope. PI stains all dead cells, while Annexin V stains those that have undergone cell death through the apoptotic pathway. The results were evaluated by comparison with three negative controls: cells transfected with DRACO only, cells transfected with dsRNA only, and non-treated cells.

##### In the experiment using HEK293 cells, many cells had detached from the wells. This may have affected the results, and therefore no conclusion could be drawn as to whether the effect of DRACO was observed.

##### From these results, in DF1 cells, co-transfection of DRACO and dsRNA resulted in higher fractions of PI-positive and Annexin V-positive cells compared to cells transfected with DRACO alone or non-treated cells, suggesting that apoptosis may be induced by the DRACO system. However, since the highest ratios were observed in cells transfected with dsRNA alone, it is possible that the dsRNA itself exerts cytotoxic effects on the cells.

##### >>Generation of cell lines

##### In verifying COCCO, the low transfection efficiency of DF-1 cells became a problem. Since avian influenza viruses can basically replicate only in chicken cells, DF-1 cells had to be used for proof-of-concept experiments involving avian influenza virus. However, if the transfection efficiency is low, the plasmid introduction efficiency will also be low, and even if our COCCO can efficiently induce cell death, the number of apoptosis events occurring within the cell population will be small. Considering that transfection itself can induce cell death, it would be ideal to create a stable cell line that constitutively expresses COCCO, so that all cells in the population express COCCO.

##### For this purpose, after transfecting the plasmids into HEK293 and DF-1 cells, selection with neomycin was performed to create stable cell lines. This was because all COCCO and negative control plasmids contained a neomycin resistance gene. Normally, after screening with neomycin resistance, single cells are selected and cloned to obtain pure stable transformants. However, since this experiment was conducted in two months, there was not enough time for cloning. Therefore, we decided to conduct subsequent experiments using cell lines in which various stable transformants were mixed.

##### To establish stable cell lines, we used pTwist CMV Neo and designed plasmids that express the following three COCCO proteins under the control of the CMV promoter.

* RIG-I-CARDapaf1 (fig32-1）
* PKR-CARDapaf1 (fig32-2)
* PKR-ΔCaspase9 (fig32-3)

![](https://static.igem.wiki/teams/5910/results/img32-1.avif)

*Figure32-1. Domain architecture of RIG-I-CARDapaf1*

![](https://static.igem.wiki/teams/5910/results/img32-2.avif)

*Figure32-2. Domain architecture of PKR-CARDapaf1*

![](https://static.igem.wiki/teams/5910/results/img32-3.avif)

*Figure32-3. Domain architecture of PKR-ΔCaspase9*

##### In addition, as negative controls, we designed plasmids expressing the following four proteins. We also created a stable cell line transfected with the vector alone, without insertion of any gene encoding the proteins to be expressed.

* non CARD RIG-I (fig33-1)
* CARDapaf1 (fig33-2)
* PKR (fig33-3)
* ΔCaspase9 (fig33-4)
* vector only

![](https://static.igem.wiki/teams/5910/results/img33-1.avif)

*Figure33. Domain architecture of non-CARD RIG-I*

![](https://static.igem.wiki/teams/5910/results/img33-2.avif)

*Figure34. Domain architecture of CARDapaf1*

![](https://static.igem.wiki/teams/5910/results/img33-3.avif)

*Figure35. Domain architecture of PKR*

![](https://static.igem.wiki/teams/5910/results/img33-4.avif)

*Figure36. Domain architecture of ΔCaspase9*

![](https://static.igem.wiki/teams/5910/results/img34.avif)

*Figure37. DF-1 stable cell line expressing RIG-I-CARDapaf1*

##### We confirmed that the desired stable cell lines had been established. In subsequent experiments, all observations were performed using these stable cell lines.

##### >>Verification of protein expression in stable cell lines

##### The expression of the transgenes was confirmed by Western blotting using an anti-Flag antibody. In the HEK293 stable cell lines, recombinant expression was detected except for CARDapaf1 and PKR-ΔCaspase9, whereas in DF-1 cells, expression was not observed except for PKR-CARDapaf1. As shown below, expression of the target protein was not detected in some samples. Therefore, subsequent results focus exclusively on those with confirmed expression. As a control for Western blotting, an anti-β-actin antibody was used to determine the amount of cells, and similar intensity bands were observed in both HEK293 and DF-1 cells.

![](https://static.igem.wiki/teams/5910/results/img39.avif)

*Figure38. Result of Western blotting of stable cell line*

*The left figure shows a Western blot using an anti-Flag antibody, and the right figure shows a Western blot using an anti-β-actin antibody.*

##### >>Optimization of dsRNA concentration

##### In the previous DRACO reproduction experiment, transfection with dsRNA alone induced a certain level of cell death, which obscured the cell death-inducing effect of DRACO. Therefore, we first examined the concentration of dsRNA to determine the optimal conditions for the assay.

##### In the HEK293 stable cell line that was generated, dsRNA was transfected at concentrations ranging from 25 ng/well to 250 ng/well. FuGENE 6 Reagent was used as the transfection reagent.

![](https://static.igem.wiki/teams/5910/results/img35-1.avif)
![](https://static.igem.wiki/teams/5910/results/img35-2.avif)

*Figure39. Result of dsRNA optimization using stable cell line*

*For the stable cell lines expressing COCCO and their negative controls, the fraction of PI-positive cells and the fraction of Annexin V-positive cells were determined for each concentration of transfected dsRNA.*

* A, Fraction of PI-positive cells in wild-type HEK293 cells (not a stable cell line) transfected with each concentration of dsRNA.
* B, Fraction of Annexin V-positive cells in wild-type HEK293 cells (not a stable cell line) transfected with each concentration of dsRNA.
* C, Fraction of PI-positive cells in HEK293 stable cell line expressing RIG-I-CARDapaf1 transfected with each concentration of dsRNA.
* D, Fraction of Annexin V-positive cells in HEK293 stable cell line expressing RIG-I-CARDapaf1 transfected with each concentration of dsRNA.
* E, Fraction of PI-positive cells in HEK293 stable cell line expressing PKR-CARDapaf1 transfected with each concentration of dsRNA.
* F, Fraction of Annexin V-positive cells in HEK293 stable cell line expressing PKR-CARDapaf1 transfected with each concentration of dsRNA.

##### For the analysis, we assumed that the fluorescence intensity per cell area would separate into positive and negative populations. We created violin plots of the fluorescence intensity for each cell and used the local minima in the distribution, which were considered to represent the boundary between the positive and negative populations, as the threshold.

![](https://static.igem.wiki/teams/5910/results/img36.avif)

*Figure40. Fluorescence expression per unit area and minimal values by cell type*

*Fluorescence intensity of Annexin and PI per cell area and the local minima in the distribution on the violin plots. For DF-1 cells, a clear distinction between the positive and negative populations could not be observed; however, for convenience, the smallest local minimum was used as the threshold.*

##### Our objective was to identify a dsRNA dosage that activates COCCO while maintaining the viability of wild-type cells. However, the unexpected death of wild-type cells, including non-treated controls, made it difficult to determine the optimal condition. Due to time constraints precluding a repeat experiment, we fixed the dsRNA concentration at 150 ng/well based on the available data.

##### >>dsRNA transfection in DF-1 stable cell lines

##### dsRNA transfection at 150 ng/well was performed on the DF-1 stable cell lines that were generated: PKR-CARDapaf1, Vector only, and on wild-type cells. (Note: This experiment was conducted before the dsRNA concentration optimization experiment described above, so a higher concentration than the optimal was used.)

##### The results were observed 24 hours later. The results are shown in the following graph.

![](https://static.igem.wiki/teams/5910/results/img-38.avif)

*Figure41. Result of dsRNA transfection in DF-1 stable cell lines*

Annexin expression levels upon transfection of 150 ng/well dsRNA in DF-1 stable cell line expressing PKR-CARDapaf1, in positive control (wild-type cells + sodium azide) and in negative controls (vector only and wild-type cells).

##### The graphs indicate that dsRNA transfection induced higher levels of apoptosis in the PKR-CARDapaf1 stable cell line compared to the control construct. This provides the first evidence that at least one COCCO construct can induce cell death in a dsRNA-dependent manner. Results for the other COCCO constructs remain inconclusive.

##### Although no statistically significant difference was observed, significance might have been detected with a larger sample size or an optimized dsRNA concentration. Additionally, since this experiment used a mixed cell population, apoptosis induction could be more pronounced if a monoclonal cell line were used.

## 2. Infection Experiment

![](https://static.igem.wiki/teams/5910/results/fig30re.avif)

*Figure42. Infection experiment diagram*

##### main achievements

* DF-1 stable cell lines expressing COCCO and negative controls were infected with influenza virus (H3N8) and analyzed using FACS.
* HEK293 stable cell lines expressing COCCO and negative controls were infected with influenza virus (H3N2) and analyzed using fluorescence microscopy.

**view details**

#### Infection experiment in DF-1 cells

##### >>FACS analysis

#####

![](https://static.igem.wiki/teams/5910/results/img43-kaitei.avif)

*Figure43. Result of FACS analysis at 6 hours post infection (n=3)*

##### We investigated whether stable cell lines expressing COCCO proteins induce apoptosis upon viral infection. Specifically, DF-1 stable cell lines were infected with influenza virus (H3N8) at an MOI of 10, and cells at 3, 6, 9, 12, and 14 hours post-infection (hpi) were stained with eBioscience™ Fixable Viability Dye eFluor™ 780, a fixable dead cell stain, and Annexin V, followed by fixation with 1% formalin. After fixation, the samples were analyzed by Dr. Daiya Ohara using FACS. Cells that were negative for eBioscience™ Fixable Viability Dye eFluor™ 780 and positive for Annexin V were considered apoptotic, and the fraction of apoptotic cells was calculated. The proportion of apoptotic cells was compared between wild-type and each of stable cell lines using a t-test. The p-values were above 0.05, and no statistical significance was observed.

#### Virus titration

##### We investigated whether stable cell lines expressing COCCO proteins could suppress viral replication. Specifically, at 9 and 12 hours post-infection (hpi), viruses propagated from RIG-I-CARDapaf1, PKR-CARDapaf1, vector-only, and wild-type cells were used to infect MDCK cells in a serial dilution, and the TCID50 was measured. Comparing the PKR-CARDapaf1-expressing cell line with the wild type reveals a higher proportion of apoptotic cells in the former. However, the CARDapaf1 and PKR cell lines, for which protein expression was not confirmed, exhibited even higher rates of apoptosis.Since the CARDapaf1 and PKR lines are expected to yield results similar to the wild type or vector-only controls, this discrepancy suggests that the primary issue with this experiment is a lack of data stability.

![](https://static.igem.wiki/teams/5910/results/img-44kaitei.avif)

*Figure44. Result of titration experiments at 6 hours*

##### From the results of the graph, at both 9 hpi and 12 hpi, the COCCO-expressing cells RIG-I-CARDapaf1 and PKR-CARDapaf1 showed no significant difference in viral replication compared to the controls. It cannot be excluded that 9 hpi and 12 hpi corresponded to the early stage of infection, and measuring at later time points would have been preferable.

##### >>Infection experiments for HEK293

![](https://static.igem.wiki/teams/5910/results/img-46kaitei.avif)

*Figure45. Result of microscopic analysis at 6, 9 and 12 hours post infection

The Annexin intensity threshold was defined as described previously to determine the percentage of Annexin-positive cells, excluding 12 hpi data due to elevated overall intensities. Data with excessive PI staining due to imaging artifacts were excluded. Analysis was limited to objects between 20 and 200 µm².*

##### We investigated whether stable HEK293 cell lines expressing COCCO proteins induce apoptosis upon viral infection. Specifically, HEK293 stable cell lines were infected with influenza virus (H3N2) at an MOI of 10, and cells at 6, 9, and 12 hours post-infection (hpi) as well as non-infected controls (MOCK) were stained with Hoechst, Annexin V, and PI.

##### In the graph, the value at 9 hpi was lower than at 6 hpi, which was attributed to variability in cell numbers prior to infection due to experimental handling. Since the amount of virus per well was uniform, wells with higher cell numbers at 9 hpi had lower virus per cell compared to other time points. A lower MOI reduces infection efficiency.

##### Therefore, the data at 6 hpi and 12 hpi were primarily used for discussion of the results.

##### At 12 hpi, the proportion of cells with mean Annexin intensity above the threshold was very close to 1 regardless of cell type, indicating that most cells had reached apoptosis.

##### At 6 hpi, Compared to wild-type cells, PKR-CARDapaf1 expressing cell line showed higher proportions, suggesting that PKR-CARDapaf1expressed in these cells tend to induce apoptosis.

##### Nevertheless, data stability remains a significant concern in the current study. As several wells were unanalyzable, further optimization of the experimental conditions for infection is necessary.

#### Virus titration experiment

##### Viral titration was performed to investigate whether stable cell lines expressing COCCO proteins can suppress viral replication. Specifically, at 9 hours post-infection (hpi), viruses propagated from RIG-I-CARDapaf1, PKR-CARDapaf1, PKR-ΔCaspase9, CARDapaf1, vector-only, and wild-type cells were used to infect MDCK cells in a serial dilution, and the TCID50 was measured.

![](https://static.igem.wiki/teams/5910/results/img45.avif)

*Figure46. Result of titration experiments with HEK 293 cell line*

##### From the graph, it cannot be concluded that the COCCO-expressing cells RIG-I-CARDapaf1 and PKR-CARDapaf1 significantly suppressed viral replication compared to the control cell lines.

## Conclusion of experiments using cell lines

##### Stable cell lines were successfully established for COCCO subtypes and several control constructs in both human and chicken cells. These represent an important achievement and provide a foundation for the future improvement and development of COCCO.

##### The obtained stable cell lines show promising results, indicating that at least one COCCO construct can induce cell death in a dsRNA-dependent manner. In future experiments, we aim to investigate this result in more detail and also to accurately evaluate the functions of other COCCO subtypes.

##### Through these experiments, we optimized experimental conditions for many procedures, including viral infection, dsRNA transfection, and cell death assays, and established standardized protocols. These protocols are recorded in detail in this Wiki and will serve as a valuable resource for future iGEM teams addressing challenges related to viral infection.

# References

[1] Rider TH, Zook CE, Boettcher TL, Wick ST, Pancoast JS, Zusman BD. Broad-spectrum antiviral therapeutics. PLoS One. 2011;6(7):e22572. doi: 10.1371/journal.pone.0022572. Epub 2011 Jul 27. PMID: 21818340; PMCID: PMC3144912.

[2] Yoneyama, M., Kato, H., & Fujita, T. (2024). Physiological Functions of RIG-I-like Receptors. Immunity, 57(4), 731–751.

[3] Zachary T. Schafer, Sally Kornbluth, The Apoptosome: Physiological, Developmental, and Pathological Modes of Regulation, Developmental Cell, Volume 10, Issue 5, 2006, Pages 549-561, ISSN 1534-5807.

<https://doi.org/10.1016/j.devcel.2006.04.008>

(<https://www.sciencedirect.com/science/article/pii/S1534580706001699>)

[4] Jang, Th., Kim, S., Jeong, JH. et al. Crystal structure of caspase recruiting domain (CARD) of apoptosis repressor with CARD (ARC) and its implication in inhibition of apoptosis. Sci Rep 5, 9847 (2015).

<https://doi.org/10.1038/srep09847>

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