iGEM 2023 — Team kuas-seoul

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# Notebook

## March

👻 Week 1

```
📖 After recruiting new members, we organized an orientation session. Students enthusiastically shared their insights about synthetic biology and iGEM. Experienced members elucidated the workings of iGEM and the club for the upcoming year. Moreover, we structured a range of assignments to facilitate a deeper understanding of synthetic biology. Also, to further enhance team cohesion, we enjoyed a delightful dinner that fostered closer connections and stronger bonds. We are excited about embarking on this iGEM journey!

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha |
| --- | --- |
```

✍️ Week 2

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📖 The newly integrated KUAS team members have conducted reviews of several iGEM wikis from previous years. Based on their specific departments, they have organized crucial factors to emphasize, identified areas for improvement, and outlined novel approaches to apply in this year's iGEM project. Additionally, plans have been made to recruit additional dry lab members to enhance the project's computational and analytical aspects. The senior members have proposed potential iGEM project ideas, such as immunotherapy for tumor diagnosis and the development of diagnostic kits for pets. These ideas reflect our commitment to addressing important challenges and advancing innovative solutions within the field of synthetic biology.

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha |
| --- | --- |
```

🫶 Week 3

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📖 Two major topics were selected for potential iGEM projects — diagnostic kits for pets, and tumor state conversion for integrated diagnosis and treatment. The significant challenge in both endeavors was the selection of a suitable target disease for diagnostic purposes. Determining an efficient diagnostic approach and tool was another obstacle. Our aspiration was to devise a method that is not only convenient but allows for direct observation with the naked eye. Although diverse topics were considered, our primary goal was to launch a project that could provide benefits to society.

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha |
| --- | --- |
```

🙇‍♂️ Week 4

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📖 Two additional members joined KUAS, bringing us great enthusiasm and further dedication to the ongoing project. To facilitate effective communication within our group, we have actively incorporated a variety of media platforms such as blog, Asana, and Slack. We also initiated our involvement in the ‘Club Activation Project’ to raise funds and extend our reach within the science community. Enhancing community engagement and cultivating interest in synthetic biology was one of our goals. We explored different strategies and activities to share our knowledge and passion with a wider audience. Furthermore, we actively engaged in brainstorming sessions to develop a comprehensive research proposal, seeking advice and suggestions to refine concepts and ideas.

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu, Jihee |
| --- | --- |

```

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## April

✍️ Week 1

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 📖 Additional brainstorming sessions were organized to explore innovative concepts with a primary focus on the integration of synthetic biology into our project.. Accordingly, we conducted an extensive study to thoroughly examine the precise definition, objectives, and varied applications of synthetic biology. Furthermore, we aimed to better understand iGEM's mission and objectives. The human practice team devised various potential activities, such as exploring collaboration opportunities with KUSSOM and other companies. We pursued opportunities for social engagement initiatives in collaboration with academic and corporate bodies to augment societal involvement and foster sustainable impact.

    - KUSSOM: Institute for Sustainability to support systematic and professional social contribution activities of school clubs.
![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/April_Week_1.jpg?ref_type=heads)

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

🤓 Week 2~4

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📖 Today was a big day! We completed the registration on iGEM! We are excited to join the iGEM community! To seek innovative ideas and build a strong foundation for our project, we conducted an extensive review of academic papers and related materials. We decided to utilize Notion for communication and efficient collaboration. Diverse applications will enhance our team's productivity toward successful iGEM!

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

## May

🗯️ Week 1

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📖 Three critical concepts were considered during topic selection. First, we assessed the potential of generating observable and significant changes via the chosen topic. Second, we evaluated the feasibility of investigating using student-level experiments [availablity]. Lastly, the topic's plausibility was a key consideration. 'Cell Free Expression system' was mentioned. We tried to analyze the concept from different aspects. For instance, protein production methods, gene circuit design applications, and others. Moreover, we reviewed relevant case studies and past iGEM projects utilizing the 'Cell-Free Expression system'. This process helped us to gain a better understanding of its implications.

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

😛 Week 2

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📖 After engaging in a comprehensive and thoughtful discussion, we decided to focus our project on 'Diagnostics' for this year. We considered various subtopics within this domain, such as tumor state determination and conversion, utilization of riboswitch-aptamers, and antibodies exhibition were some of the examples. Further, we divided into four groups to research a different facet of these diagnostic applications. During the research process, we were surprised by the immense potential that synthetic biology holds in the realm of diagnostics. This realization expanded our perspectives. We look forward to the future of synthetic biology and its potential to significantly impact a broader range of fields.

| Participants | Minjung, Minju Ha, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

✍️ Week 3

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📖 Four different ideas were developed.

    1. Diagnosis and Treatment for Myzus persicaee
    - Problem: Myzus persicae bring disease to potatoes and spread fast. Not all kinds of aphids are harmful, a specific species Myzus persicae causes the problem.
    - Solution: Create a kit to detect Myzus persicae and alarm farmers

        a) Detect and utilize the specific protein in Myzus persicae's saliva

        b) Utilize aphid cuticular proteins

        c) Feed Myzus persicae-modified protein to exhibit a certain color

    2. Diagnositic for Feline Panleukopenia Virus
    - Problem: No treatment for the virus, Kill cats; only vaccination is available but its effect is limited.
    - Developed Solution: Diagnosis and Treatment of Feline Panleukopenia Virus

        a) Cell-free system: Select and analyze Target seq (FeLV virus_ p27 antigen concentration)

        b) P27 antigen-specific binding Antibody+Fluorescence

    3. Phage therapy
    - Antibiotic resistance and phage therapy
    - Utilizing site-specific recombination
    - Problem: Mutation

    4. Biosensor for Ovarian Cancer Early diagnosis
    - Cell-free system-based color expression, Paper-Based Analytical Device
    - Possible biomarkers: CA-125

    ---

     We revised all different project ideas and shared the advantages and limitations of the suggested projects. Furthermore, we made preparations for the Creative Challenge Program (CCP). This year's goal of the CCP was about ESG. We proceeded to create a detailed action plan and allocated responsibilities for fundraising.

| Participants | Minjung, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

✍️ Week 4

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📖 Following more discussions and a voting process, we narrowed down our options to two topics out of the initial four discussed during last week's meeting. Subsequently, we prepared two concise versions of the Promega Impact Grant proposal for these shortlisted topics. Our objective was to evaluate both topics from diverse perspectives, and the questions outlined in the Promega Impact Grant guidelines greatly aided us in reaching a decision.

    Afterward, to finalize our project topic and prepare for the Promega Impact Grant application, we conducted another round of voting to determine the most desirable and interesting topic. Ultimately, we selected the 'Cervical Cancer Diagnostic Pad' as our project topic.

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu |
| --- | --- |
```

✍️ Week 5

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📖 Based on the selected topic, we endeavored to refine and enhance the project ideas to ensure their validity and effectiveness. This week, we focused on fundraising to gather sufficient resources for further investigations and experiments. We explored various methods beyond fundraising to generate the necessary resources. Additionally, we devised a plan to engage the public through activities such as a mentoring program, seminars targeting various audiences, collaborations with other iGEM teams, and utilizing social media platforms. Particularly, members of the Human Practice team made significant efforts to reach out to companies and organizations to implement these plans.

| Participants | Minjung, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

## June

✍️ Week 1

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📖 We collectively revised the Impact Grant form, meticulously examining each question to grasp its purpose and intricacies. This process enabled us to evaluate the limitations and devise strategies to refine our project ideas accordingly. Simultaneously, we clarified the importance and potential impact of our project.

    In essence, our project is focused on designing a sanitary pad embedded with a biosensor capable of detecting cervical cancer through visual indicators. The concept leverages the widespread accessibility and convenience of sanitary pads, introducing a host of benefits on a local and global scale. The innovation facilitates early-stage diagnosis of cervical cancer through a simple, cost-effective process, significantly contributing to women's health worldwide.

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

✏️ Week 4

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📖 Our team completed the initial draft of the project description this week, focusing on potential biomarkers for cervical cancer that can be detected through analysis of vaginal fluid. After conducting thorough research, we identified ACTN-4 as a promising biomarker for further investigation. Our primary objective was to explore the potential of utilizing ACTN-4 as a diagnostic tool in cervical cancer detection.

To achieve this goal, we delved into the application of ACTN-4 within a Riboswitch-Aptamer system. The aim was to translate ACTN-4 utilizing a cell-free system and detect it using a specific aptamer. In order to effectively implement this methodology, we conducted a detailed investigation into identifying aptamers that can specifically bind with the target protein, ACTN-4.

Furthermore, our team sought to expand our project's reach and impact by enhancing our fundraising efforts. To achieve this, we submitted applications to fundraising platforms such as WADIZ and Kakao Corp., aiming to gather the necessary resources for the successful execution of our research and development activities.

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/June_Week_4-1.jpeg?ref_type=heads)

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/June_Week_4-2.png?ref_type=heads)

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

## July

🗯️ Week 1

```
 📖 Various innovative methodologies have been proposed for our project:

1. Anti-ACTN aptamer
- The specific sequence of the aptamer has not been disclosed. Although Uniprot lacks aptamer information, the target information is available. SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and protein-protein interactions can aid in aptamer creation and modification. Exploring similarities in entry2 could enhance binding affinity and aptamer modification.

2. Targeting SEB
- Recognition: SEB2
- Proposed Alterations:
- Structural alteration of the aptamer to induce a color change from red to purple.
- The aptamer binds both to gold nanoparticles and the target. Salt is required, potentially inducing ion exchange. High target specificity is crucial for the aptamer's effectiveness.

3. Using Antibodies: actinin-4 mRNA
- Approach:
- Targeting actinin-4 mRNA
- Requires primer and nucleic acid probe
- Induce binding using only antibody fragments instead of using the whole antibody.
- Since the secondary antibody amplifies the signal, designing the antibody to bind to both the invariant and variable regions might enable signal amplification. Designing the variable region could be synthetically utilizable.

In addition to the proposed methodologies, the integration of a two-component system and dimerization regulation has been suggested to enhance the validity of the circuit. To enhance the circuit's validity, further research on the riboswitch system and aptamers was needed. Given time constraints, it was also essential to explore efficient alternatives to SELEX for implementation. These methodologies encompass a broad spectrum of techniques, each offering unique potential for our project. Further exploration and validation will steer us towards the most effective diagnostic tool for cervical cancer detection.

| Participants | Minjung, Minju Ha, Jeongin, Sungeun, Minji, Ayoung, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

🍀 Week 2

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📖 There was doubt regarding the validity of utilizing ACTN4 as a biomarker due to the low incidence of patients with diseases caused by ACTN4. Many patients were observed to have mutations in different genes. We came together to discuss and debate this issue.

 We also talked about constructing a genetic circuit using aptamers. The convenient diagnostic process through color changes was a crucial aspect of this project. Various ideas were proposed, including attaching gold nanoparticles to aptamers to observe color changes, expressing color proteins, or utilizing pH indicators to create color changes. In order to incorporate synthetic biology, we tried to regulate the transcription and translation process by genetic circuit modification.

In the Dry lab, we investigated the probability of gene expression data occurring when aptamers bind to the set targets. We also explored processes to deduce mechanisms for aptamer-protein binding and researched programs to predict gene expression.

 Moreover, we outlined volunteer projects to target different audiences. For high school students, we proposed educational programs and experiments on gram-positive and gram-negative bacteria. We also worked on programs emphasizing laboratory safety and explaining synthetic biology. We planned interactive activities for young children such as drawing a cell factory, creating a simple introduction to synthetic biology through a storybook, and making a DNA structure.

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/July_Week_2-1.jpeg?ref_type=heads)

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/July_Week_2-2.png?ref_type=heads)

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

🖥️ Week 3

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📖 In the Dry Lab, we discussed the selection of aptamer candidates using the PPAI program, initially narrowing down to about 10-20 candidates. After further narrowing down the selection from a random library via wet lab experiments, we expected to re-run the PPAI program to finalize the aptamer candidate pool.
    Additionally, we discussed implementing a biosensor Arduino system. The idea that a Biosensor Arduino system can be applied for prokaryotic protein expression was interesting. Furthermore, considering coding that would enable varying levels of luminescence based on the progression of cancer could potentially enhance the diagnostic utility of the kit.

    We also discussed seeking further advice on the SELEX technique and experiments. Consulting with our professor helped to increase the feasibility and validity of the project. Due to the challenge of conducting SELEX within a short timeframe and limited experimental resources, we explored alternative methods. Consequently, we devised strategies such as manipulating the receptor-binding component and utilizing the EGF receptor instead of an aptamer.

    Furthermore, we contemplated the practicality of applying the project to cervical cancer detection by integrating it into a real-life application like a sanitary pad.

    In order to refine the project idea, we extensively researched numerous papers and materials. Throughout this process, we acquired a wealth of knowledge and found great satisfaction in observing the project's development.

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/July_Week_3.jpeg?ref_type=heads)

| Participants | Minjung, Minju Ha, Jeongin, Sungeun, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu |
| --- | --- |
```

🫤 Week 4

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📖 Additional biomarkers were proposed aside from ACTN-4. The HRP sequence was put forward, but its lengthy gene sequence poses challenges for integration into the reporter gene. Chromoprotein was also recommended due to its ability to display distinct colors easily visible to the naked eye. However, codon optimization would be necessary for practical application. Moreover, HMGB1, HPV-07, and C-9S were discussed as potential biomarkers.

    The selection of biomarkers was a crucial consideration. We aimed not only to focus on a biomarker's intended function but also on its unique aspects such as expression patterns, activities, and alterations under varying conditions.

---

    In the dry lab, we employed the HDock program to generate an aptamer library for various biomarkers. This software also assisted in predicting protein interactions with DNA/RNA molecules by utilizing a combination of two distinct methods. The first method utilizes existing templates as a guide, while the second method searches for the optimal docking configurations. Analyzing the docking score and confidence score assisted in estimating the likelihood of binding.
![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/July_Week_4.png?ref_type=heads)

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu, Jihee |
| --- | --- |
| HDock | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu, Jihee |
```

## August

✍️ Week 1

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📖 We have established a funding plan through WADIZ. We also have devised reward schemes for donors and supporters. Furthermore, we have purchased the necessary materials and prepared presentations for students participating in our education programs and campus tours.

    To progress with the project, we looked for various tools, such as RNA aptamer generation and aptamer prediction models. We also contemplated the utilization of diverse devices, including CLSM, to facilitate the experiment. Additionally, we made efforts to reach out and secure sponsorships from various companies and organizations for our project. Moreover, many chromoproteins were suggested for target protein color expressions, such as amilCP, gfasPurple, and arBlue.

    The contributions of everyone from their respective affiliations to the project were significant. The collaborative effort of everyone working towards a common goal was truly meaningful.

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Minji, Ayoung, Sangyeon, Seunghun, Hyesu, Jihee |
| --- | --- |
```

✏️ Week 2

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📖 ACTN4 was selected as the target protein. More studies were conducted to understand the characteristics of the ACTN4 protein, such as length, repeats, amino acids, and other essential aspects. Additionally, we delved into methodologies and their effects on expressing ACTN4 through cell-free expression. In addition, we initiated gene circuit design, incorporating various vectors, promoters, and polymerases.

After protein expression, confirmation experiments will be required to ensure successful expression. For the selected aptamer, conducting a binding affinity test is essential. Hence, we explored various methodologies to validate these concepts.

Furthermore, E6 and E7 were suggested, each with its own specific RNA aptamer for binding.

We conducted further investigations into the biosensor application. The LFA assay was proposed, considering both sandwich and non-competitive methods.

---

This week, we visited a high school and conducted various programs to ignite interest in synthetic biology. We prepared simple experiments and engaged in debates about how synthetic biology can be applied in different domains. We thoroughly enjoyed this activity, and the enthusiastic attitude displayed by the students truly made our day!

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/August_Week_2.jpeg?ref_type=heads)

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

👏 Week 3

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📖 The aptamer for ACTN 4 proved to be excessively expensive, surpassing our budget constraints. Consequently, due to economic considerations, we had to explore alternative biomarkers for our project. PTK7 and CEA were proposed as viable replacements. Among the potential target proteins, we chose the CEA N domain to reduce translation time and protein ordering costs by focusing on a shorter protein length.

    Using software like OpenVector Editor and SnapGene, we proceeded to design the plasmid. We also ordered the template DNA required for our experiment.

    Expanding beyond the protein's role as a biomarker for diagnostic purposes, we evaluated the project from various perspectives, including its economic implications when applied in real-life scenarios.

    Furthermore, we extensively researched the most suitable aptamer for the CEA N domain. We not only assessed its binding affinity based on existing literature but also employed computational analysis in the dry lab. To observe the binding between the aptamer and protein, we planned an EMSA experiment.

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/August_Week_3.jpeg?ref_type=heads)

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

👟 Week 4

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📖 We explored various EMSA protocols, researching fundamental EMSA principles, available EMSA kits, and methods that do not require kits. To enhance visibility, we considered incorporating FAM-tag and biotin tags. For safety concerns, we tried to look into kits that do not use biotin or radioactive substances, seeking a safer alternative for EMSA.

    During this week, we successfully ordered all the necessary materials for our experiments, including template DNA and aptamers. We are really excited to embark on this experimental journey!

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Minju Cha, Hyesu, Jihee |
| --- | --- |
```

📹 Week 5

```
📖 This week, we focused on preparing for our promotion video, including drafting the script and creating subtitles in multiple languages to reach a wider audience. Additionally, we worked on the judging form for iGEM.

    Simultaneously, we contacted various organizations for our human practice activities, particularly our Community Collaboration and Participation (CCP) initiatives.

    Furthermore, we organized the order of all wet lab experiments and underscored the importance of validating our project concepts. For instance, we prioritized tasks like protein expression via CFE, SDS-PAGE, protein purification, EMSA, PAD, and more.

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Seunghun, Minju Cha, Jihee |
| --- | --- |
```

### # 🧪Experiment Week [September-October]

## September

🤩 Week 1

```
📖 For the project, we generated ideas for the project and product names, considering a variety of options. Ultimately, we decided on 'CEApture' as our project name. This choice incorporates the name of our target protein, the CEA N domain, and the utilization of aptamers, and signifies our focus on capturing the CEA N protein for the diagnosis of cervical cancer.

    Furthermore, we initiated the preparations for our wiki page by outlining the elements to be emphasized. We strategized and brainstormed ways to make our ideas stand out and ensure that our written content and figures were clear and easily understandable. We really concentrated on this because we realized that effective communication and the dissemination of our ideas within the community are also important.

---

    To design our aptamer for specific binding to the CEA N domain protein, we used EFBALite. This allowed us to expedite the otherwise intricate and time-consuming SELEX process. We also enhanced the codes and developed a version tailored for our project. The program's importance lies in its ability to illustrate the binding interaction between the aptamer and the protein.

---

    We organized a campus tour for high school students, aiming to provide them with inspiration and motivation. Witnessing their happiness and enthusiasm during the tour was truly enjoyable and inspiring for us.

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/September_Week_1.jpeg?ref_type=heads)

| Participants | Minjung, Minju Ha, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Minju Cha, Hyesu, Jihee |
| --- | --- |
| EFVALite | Sangyeon |
| Volunteer | Sujin, Minji, Ayoung, Jeongin, Sungeun, Hyesu |
```

✍️ Week 2

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📖 Different wiki pages were assigned to each member, and they prepared draft content for their respective sections on the actual wiki page. Additionally, some members outlined the figures they intended to insert for better understanding. This outlining process greatly assisted in thinking about the project and organizing how we plan to share it with others.

---

    H-NMR spectroscopy and PAD were performed to analyze the aptamer-protein interaction. Due to the unstable peaks shown, Analyzing the data posed an additional challenge.

---

    MATLAB coding was employed for quantitative visualization of the aptamer-protein binding affinity. Specifically, we utilized this approach for mathematical estimation of the binding affinity between the aptamer N56 and our target protein, CEA N. This was demonstrated through the calculation of the Kd value.

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/September_Week_2-2.jpeg?ref_type=heads)

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/September_Week_2.jpeg?ref_type=heads)

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu, Jihee |
| --- | --- |
| H-NMR, PAD | Seunghun |
| MATLAB | Minju Ha |
```

👊 Week 3

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📖 The Human Practice team organized a 'Synthetic Biology Debate' to enhance public engagement. We made progress in scheduling, promoting the event, and coordinating related activities. Our team member, Sungeun Lee, actively participated in the event and emerged as the winner of the Synthetic Biology Debate Competition. This event not only aided in securing resources for our project but also presented a valuable opportunity to engage the public with biology in an intriguing manner.

---

Other team members concentrated on preparing for the presentation video. We allocated a sufficient amount of time to each part of our iGEM activities. To ensure a well-structured format, we engaged in detailed discussions. Furthermore, we made an effort to view our scripts from the audience's perspective to confirm that our delivery is effective.

---

The binding affinity prediction model was required to calculate how effectively our aptamer binds to CEA. Thus, the BERT model was considered to directly calculate protein binding affinity. Ultimately, diverse software will be utilized to assess the DNA's core part and aptamer affinity.

---

Through H-NMR Spectroscopy Experiment for N56 and H-NMR Spectroscopy and PAD Experiment for CEA Protein, primary Imaging results were obtained.

---

We proceeded with the cell-free expression of our CEA N domain template gene. The preparation of buffers was essential. Additionally, we conducted a protein expression test via SDS-PAGE. As a contingency, in case the cell-free expression system proves unsuitable for our target protein, we prepared for a protein expression test via transformations. We performed the transformations, further harvested cells, and conducted a mini-prep. We also delved deeper into native PAGE to explore alternatives to the EMSA kit. This involved a thorough study of the incubation method for protein and aptamer, which took more time than anticipated. This meticulous process included scrutinizing and precisely preparing all the buffers and components step by step.

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/September_Week_3-1.jpeg?ref_type=heads)

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/September_Week_3-2.jpeg?ref_type=heads)

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu, Jihee |
| --- | --- |
| Human Practice | Minji, Ayoung, Sungeun |
| BERT Model | Sangyeon |
| H-NMR | Seunghun |
| Experiment | Sungeun, Minju Cha, Jeongin, Minjung, Minji, Minju Ha |
```

🍀 Week 4

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📖 The Human Practice team created informative card news to educate about experiment safety and synthetic biology, which was shared via social media. Additionally, various methods were employed to raise awareness about cervical cancer. The card news proved effective, utilizing diagrams, pictures, and simple explanations to efficiently convey different topics.

---

The dry lab team also commenced the design of our wiki page. We determined the theme and color scheme and began designing subpages. To facilitate better communication and gather suggestions, we incorporated suggestion boxes within Notion. We are eager to showcase our progress and contributions for this year's iGEM on our Wiki!

---

We cultivated E. coli at different temperatures for varying durations to optimize the experiment. The experiment was carried out at 37°C, 30°C, and 16°C. For purification, we explored different methodologies. In our pursuit of EMSA, we followed the entire process under native conditions, avoiding the use of detergents. Furthermore, we initiated additional reactions for cell-free expression by extracting plasmids from transformation. This provided valuable data for comparing protein expression within the cellular system and the cell-free system. The transformation process was particularly effective, enabling an increase in protein concentration. Numerous SDS PAGE experiments were conducted to verify protein expression.

We further proceed with protein purification by utilizing 2 different resins. We applied Ni-NTA resins and cobalt resins for comparisons. We utilized these beads since they are effective in purifying His-tagged proteins.  Each type of bead has its advantages and limitations. Ni-NTA beads have higher availability, cost-effectiveness, and versatility. Cobalt resins, on the other hand, enhance selectivity but are more expensive. The contamination was the most crucial part of the entire experiment. In order to minimize the possibility of contamination, we tried to autoclave all the equipment we were going to utilize for the purification process. Additionally, we recreated a protocol for protein purification. Such as imidazole concentration was regulated and additive agents (PMSF, Benzonase Endonuclease, etc) were selected. Also, in order to confirm the purification process was successful, we again utilized SDS PAGE as confirmation.

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/September_Week_4-1.jpeg?ref_type=heads)

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/September_Week_4-2.png?ref_type=heads)

| Human Practice | Minji, Ayoung |
| --- | --- |
| Dry Lab | Minju Ha, Sangyeon, Hyesu, Jihee |
| Experiment | Sungeun, Sujin, Jeongin, Minju Cha |
| Lab Assistant | Minji, Minjung, Minju Ha |
```

## October

✍️ Week 1~2

```
📖 Another round of protein purification was conducted. To ensure precision and accuracy in the purification process, we standardized the concentration of all the samples using a nanodrop. Specifically, we collected samples including unbound protein, wash samples 1, 2, and 3, and elution buffer samples at varying concentrations (100mM, 250mM, 500mM). We obtained a total of four types of comparable samples: those grown at 30°C and purified using Ni-NTA beads, those grown at 37°C and purified using cobalt resin, those grown at 37°C and purified using Ni-TA resin, and samples from the cell-free system purified using Ni-NTA resin. We compared the results and selected the well-expressed, purified samples for EMSA experiments. Unfortunately, we faced limitations due to an insufficient quantity of designed aptamers. Therefore, we selected the most well-purified protein for actual EMSA experiments. We proceeded with EMSA to observe the aptamer-protein binding interaction. This was done within the native page to maintain protein structures. Due to time constraints, we were unable to proceed with the LFA assay for practical application. However, diverse wet lab and dry lab experiments helped confirm the CEA N domain protein-N56 aptamer-specific binding. This further indicated the effective utilization of sanitary pads and the potential for commercialization.

---

    All the wiki pages were written and revised by each member. With the assistance of our Wiki designer, we dedicated a significant amount of effort to designing our own pages. We collaborated and supported each other in writing and creating figures to enhance the accessibility and quality of the content.

---

    Wet lab experiments, dry lab modeling, and human practice activities were all completed this week. We are so glad that we've made it on our iGEM journey! This iGEM experience has not only provided us with valuable learning opportunities and academic growth, but it has also illuminated our educational path. Thank you to everyone for being a part of this incredible experience!

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/October_Week_1.jpeg?ref_type=heads)

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/October_Week_1.png?ref_type=heads)

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/October_Week_2.jpeg?ref_type=heads)

| Participants | Minjung, Minju Ha, Sujin, Jeongin, Sungeun, Minji, Ayoung, Sangyeon, Seunghun, Minju Cha, Hyesu, Jihee |
| --- | --- |
| Experiment | Sungeun, Sujin, Jeongin, Minju Cha |
| Lab Assistant | Minjung, Sangyeon |
| Wiki | Hyesu |
```

![](https://gitlab.igem.org/2023/kuas-seoul/-/raw/main/image/Notebook%20Image/%ED%86%A0%EA%B8%80%EC%97%90_%EC%95%88%EB%84%A3%EA%B3%A0_%EB%A7%88%EC%A7%80%EB%A7%89_%EB%A7%A8_%EB%B0%91%EC%97%90_%EC%99%84%EC%A0%84_%EB%A7%88%EB%AC%B4%EB%A6%AC.png?ref_type=heads)

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Establishing the logical flow of an experiment with DBTL process{% endblock %}
{% block page\_content %}

## [DBTL, synthetic biology engineering]

Our main concept idea is to create a simple, everyday-use, built-in cervical cancer diagnostic kit within sanitary pads. To realize this idea, we have gone through multiple Design-Build-Test-Learn (DBTL) cycles during the process of designing our idea and a synthetic biological pathway.

### [Design 1: 'Finding Cervical Cancer Biomarker']

---

Our goal was to diagnose cervical cancer using menstrual blood, which led us to select ACTN4, detectable in cervical vaginal fluid, as a biomarker for cervical cancer [1].

The primary cause of cervical cancer is due to the infection of the Human Papillomavirus. We have also set E6 and E7, proteins of the Human Papillomavirus as another biomarker for cervical cancer diagnosis [2].

### [Build 1: 'Organizing Synthetic Pathway']

---

###### Idea 1

We selected ACTN4 and E7 as biomarkers for cervical cancer. When ACTN4 or E7 binds with RNA Aptamer, the Aptamer riboswitch is activated, causing the RBS site on mRNA to open, leading to the expression of fluorescent proteins.

idea 1 sketch
![<FIgure 1. idea 1 sketch>](https://static.igem.wiki/teams/4953/wiki/engineering1-1.png)

###### Idea 2

When ACTN4 or E7 binds with RNA Aptamer, the Aptamer riboswitch is activated, causing the RBS site on mRNA to open. This leads to the expression of a transcription factor (TF), which in turn triggers the expression of chromoproteins from an additional plasmid we introduced.

idea 2 sketch ![<FIgure 2. idea 2 sketch>](https://static.igem.wiki/teams/4953/wiki/engineering1-2.png)

### Test1: 'Trying to find aptamer sequence & signal protein

---

To discover a new DNA Aptamer sequence that binds to ACTN4 and E7, the KUAS team's Dry lab utilized a program called EFBALite (software of Vilnius-Lithuania, 2021 iGEM team) to conduct in-silico analysis. [AI model updates](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27aimodelupdates%27%29%20%7D%7D)

### Learning 1:'Troubleshooting'

---

###### Idea 1 & 2 Problems:

The DNA Aptamers found for ACTN4 and E7 proteins using the EFBALite software (from the Vilnius-Lithuania, 2021 iGEM team) were as follows: 'GTACTACCCCCCCCC' and 'GTTTTTTTTT.'

The sequences generated initially exhibited a high degree of monotony. Sangyeon Lee from KUAS-Dry Lab mentioned,

*"ACTN4 has a complex structure, and in situations where artificial intelligence models find it challenging to make predictions, they tend to simply opt for average or safe values to minimize errors."*

Consequently, these DNA aptamers were deemed unusable, and due to time and cost constraints, we began searching for alternative cervical cancer biomarkers and pre-existing aptamers.

###### Idea 1 & 2 Troubleshooting:

1. Search for protein biomarkers reported in the literature for cervical cancer.

2. The biomarker must be detectable in menstrual blood since it is the target source.

3. A specific aptamer for the biomarker must be in DNA form, as RNA aptamers are impractical at room temperature.

###### Idea 2 Problem

For chromoproteins, it was anticipated that they would not be easily visible in a cell-free system, which does not use a sufficient quantity of cells for visible results. Additionally, plasmid-based expression was expected to be challenging in a cell-free environment.

###### Idea 2 Troubleshooting:

We concluded that we should search for a reporter protein that is visible to the naked eye even in a cell-free system instead of chromoprotein.

>We considered the alternative of utilizing catechol (2,3)-dioxygenase (C23DO), a colorimetric enzyme, which turns yellow upon oxidizing catechol. It offers the advantage of a clear yellow color change visible to the naked eye. However, it was deemed impractical to introduce catechol into sanitary pads, making its application challenging.

### Design2: 'Another biomarker for cervical cancer and its DNA aptamer

---

###### 'Other Biomarkers':

We explored additional biomarkers that meet the three conditions mentioned earlier: CEA (Carcinoembryonic Antigen) and PTK7.

1. We found the biomarker PTK-7 and its DNA aptamer Sgc-8 [3].

2.We found the biomarker **CEA** and its DNA aptamers **N54, N56, N57, N59, N65, and N71 [4]**.

### [Build2: 'Lateral Flow Assay']

###### 'Chose Different Diagnostic Method'

We decided to use the lateral flow assay (LFA) as the diagnostic method and incorporated it into the sanitary pad.

![<Figure 3. Sanitary Pad and LFA Schematic>](https://static.igem.wiki/teams/4953/wiki/engineering1-3.png)

### [Test 2: H-DOCK "Protein-Aptamer Binding Affinity Calculation Program” ]

Among the six DNA aptamer candidates proposed in the reference literature for binding with CEA protein, KUAS-Dry Lab selected the N56 aptamer as the ideal candidate for use in the lateral flow assay (LFA). This decision was based on modeling the CEA N domain & DNA aptamer affinity using the H-DOCK program.

**[Result]**

|  | N54 | N56 | N57 | N59 | N65 | N71 | cApt |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Docking score | -219.07 | -226.66 | -218.33 | -226.18 | -219.93 | -217.00 | -221.87 |
| Confidence score | 0.7992 | 0.8225 | 0.7968 | 0.8211 | 0.8020 | 0.7925 | 0.8081 |

*Docking score*

: more negative **docking score** means a more possible binding model

*Confidence score*

: **confidence score** > 0.7: very likely to bind

0.5 < **confidence score** < 0.7: the two molecules would be possible to bind

**confidence score** < 0.5: unlikely to bind)

**N56** > **N59** > cApt > N65 > N54 > N57 > N71

###

###### 'N56 aptamr','Sqc-8'&'CEA, PTK7 protein'

Based on the results from the dry lab, we have selected the aptamer N56 with the highest confidence score, which binds to the cervical cancer biomarker CEA. Additionally, we have chosen the aptamer sequence, Sgc-8, which is publicly available in the literature, and its corresponding marker protein, PTK7, to work with.

However, for PTK7, as it is a human protein, its full gene sequence was exceedingly long, exceeding 85,000 base pairs, making it impractical for insertion into a plasmid. We attempted to identify only the domain responsible for interaction with the aptamer, but unfortunately, we couldn't confirm the interaction domain with sgc8. Consequently, we have decided not to use it.

---

## [Experiment DBTL, synthetic biology engineering]

![<Figure 4. Experimental workflow>](https://static.igem.wiki/teams/4953/wiki/engineering1-4.png)

###### ‘CEA expression plasmid design’:

We have constructed a plasmid for the expression of the CEA N domain.

This is because the N56 aptamer binds to the CEA N domain, and the full protein sequence of CEA is too long to be entirely accommodated in a plasmid. Therefore, only the CEA N domain was inserted into the plasmid.

![<Figure 5. pET-28a(+) vector with the CEA N domain gene sequence inserted.>](https://static.igem.wiki/teams/4953/wiki/engineering1-6.png)

### [Design 1: ‘Cell-Free Expression for EMSA’]

We will insert the CEA sequence into a plasmid and use E. coli extract for Cell-Free Expression to induce protein expression. Following protein expression, we will confirm the binding of our selected aptamer to CEA using the Electrophoretic Mobility Shift Assay (EMSA) method. ([Design](https://2023.igem.wiki/kuas-seoul/design)\_EMSA)

###### [Build 1: ‘Cell-free expression’]

We will insert the CEA sequence into a plasmid and use E. coli extract for Cell-Free Expression to induce protein expression. Enzymes present in the E. coli extract induce protein expression from the plasmid even in the absence of cells.

###### [Test 1 : ’Expression test’ & ‘Purification’ & ‘SDS-PAGE’]

![ Figure 6. CFE protein expression SDS-PAGE result](https://static.igem.wiki/teams/4953/wiki/engineering1-5.png )

###### [Learn 1: 'Troubleshooting']

The Cell-Free Protein Expression samples did not indicate significant protein overexpression when comparing between the negative and the samples. This was evident as the expression weight markers where CEA proteins were supposed to be expressed did not show a significant expression signal.

Furthermore, after the purification process, the SDS-PAGE result showed almost no discernible coloration in the bands, suggesting that there was minimal protein presence. Consequently, we concluded that the protein obtained through cell-free expression would not provide a sufficient quantity of protein for conducting EMSA experiments. Therefore, we decided to use cellsfor this purpose.

Additionally, during the purification process, we encountered an issue with the Ni-NTA spin column. It seemed that there was a problem with the column membrane, as during the equilibration step, even after centrifugation, the buffer did not flow down as expected. This issue led to the replacement of the Ni-NTA spin column for negative control samples. As a result, for subsequent steps, we opted to use nickel beads for protein purification to ensure a smooth and efficient process.

### [Design 2: 'Protein expression in cells for EMSA.']

We initially attempted protein expression using a cell-free system for CEA, but the protein yield was low, and the target protein was not overexpressed. Due to these issues, we decided to switch to cell-based protein expression to ensure sufficient protein production.

The chosen strain for protein expression: ***E. coli (DH5alpha\_DE3)***

1. **Versatility**: E. coli is a commonly used host organism for protein expression in research and biotechnology due to its well-characterized genetics and the availability of various expression vectors.

2. **Rapid Growth**: E. coli has a short generation time, allowing for quick production of protein.

3. **Strong Promoters**: The DH5alpha\_DE3 strain contains a T7 RNA polymerase gene under the control of the lacUV5 promoter, which can be induced by adding IPTG (Isopropyl β-D-1-thiogalactopyranoside). This system enables tight control of protein expression.

4. **High Yield**: E. coli can produce a high yield of recombinant protein when optimized for the specific protein of interest.

###### [Build 2 : '16°C cell culture’]

![ Figure 7. ](https://static.igem.wiki/teams/4953/wiki/engineering1-7.png)

1. According to Farewell and Neidhardt (1998, September), as the temperature increases, the elongation rate of protein synthesis consistently shows an upward trend. However, concerning cell growth rate, when it exceeds 37°C, the rate of increase slows down. From this, it can be inferred that excessively high temperatures may lead to the phenomenon where protein synthesis speeds up, but cell growth rate slows down, possibly indicating that ribosomes are unable to synthesize proper proteins. Therefore, when conducting cell cultivation at high temperatures, there is a risk of rapid acceleration in protein expression, potentially resulting in the aggregation of expressed proteins.

2. ‘*It is possible that all of these ribosomes are indeed functioning and producing protein but that the rate of protein degradation is greatly increased.’* Farewell, A., & Neidhardt, F. C. (1998, September) Therefore, if the CEA N domain protein is expressed too rapidly, there is a possibility that some of the expressed CEA N domain protein may degrade, resulting in lower protein purity, which may not be suitable for use in EMSA.

For these reasons, we initially cultured the cells at a lower temperature of 16 degrees Celsius to slow down protein expression and prevent aggregation.

###### [Test 2: 'Expression test']

![<Figure 8. 16°C cell culture expression test result >](https://static.igem.wiki/teams/4953/wiki/engineering1-8.png )

###### [Learn 2: ‘CEA protein expression’]

The E. coli culture temperature was too low for efficient CEA protein expression, leading to inadequate expression.

Consequently, we needed to find the optimal temperature for E. coli (DH5alpha\_DE3) to express the CEA protein. We conducted E. coli cultures at various temperatures to determine the most suitable one for CEA protein expression.

We also learned that pre-adjusting the temperature before reseeding the cells and then culturing them results in more stable cell growth.

### [Design 3: ’Protein expression in cells for EMSA.’]

######

1. At 16°C, when performing cell culture followed by reseeding and IPTG treatment for expression testing, it was evident that E. coli was too cold for efficient expression of the CEA N domain protein.

2.

‘The optimal temperature for E. coli growth is usually considered to be 35–40°C.’ Chih-Yu Yang et al. (2020)

‘The growth rate depends on the rate at which the cell is able to synthesize new proteins 6,12. This, in turn, depends on the cellular ribosome concentration, but also on how efficient each ribosome is used.’ (Bosdriesz et al., 2015)

In other words, the optimal conditions for cell growth imply having a high concentration of active ribosomes. For this cycle, we set the cell culture temperature at 37°C, which falls within the range of 35-40°C, where E. coli's cell growth rate is the highest. This temperature ensures that our target protein can be produced efficiently in an environment with a high concentration of active ribosomes.

###### [Test 3-1: ‘Expression test’]

![ <Figure 9. 37°C cell culture expression test result>](https://static.igem.wiki/teams/4953/wiki/engineering1-9.png)

###### [Build 3-2: ‘30°C cell culture’]

When conducting cell culture at 37°C, there is a risk of rapid acceleration in protein expression, potentially leading to the aggregation of expressed proteins. To mitigate this risk, we also conducted cell culture at a lower temperature of 30°C. 30°C is also a commonly used temperature for cell culture.

###### [Test 3-2: ‘Expression test’]

![<Figure 10. 30°C expression test result>](https://static.igem.wiki/teams/4953/wiki/engineering1-10.png)

###### [Learn 3-1, 2: ‘Proper Temperature for CEA protein expression’]

Overall, it appears that protein expression was more active at 37°C compared to 30°C. Despite concerns about the high temperature of 37°C potentially leading to protein aggregation during the expression process, we were fortunate **not to observe any signs of aggregation** on SDS-PAGE. Therefore, we have chosen **37°C** as the temperature at which E.coli can express CEA N domain protein vigorously without causing aggregation.

###### **[Design 4: ‘Protein purification for EMSA’]**

###### [Build 4: ‘Protein purification by Nickel bead’]

The initial purification process using Nickel beads

1. After culturing 200 ml of cells, we retrieved the cultured cells and transferred them to four 50 ml conical tubes.

2. In two separate conical tubes, we added 2.5 ml of lysis buffer each and resuspended the cells.

3. After resuspension, we combined the contents of one of the 50ml conical tubes into a single tube, resulting in a total of 5ml of lysis buffer for 100ml of cultured cells.

4. Since our lab didn't have 5ml tubes available, we divided the lysate evenly into 2ml Eppendorf tubes, with each containing 1.6ml of lysate.

5. We added 400 microliters of beads to another 2ml Eppendorf tube and centrifuged it. We discarded the supernatant, which contained the beads, and repeated the wash steps twice using the same buffer used for resuspension.

6. We added 1.6ml of cell lysate supernatant to the washed beads and shook them for 1 hour and 30 minutes.

7. As the lab didn't have a shaking machine, we adjusted an incubation device to 10°C, placed the tubes in ice, and incubated them while shaking.

8. To ensure proper mixing, we manually shook the tubes for 10 minutes.

9. After incubation, we centrifuged the tubes, discarded the supernatant (unbound part sample), added wash buffer, and immediately centrifuged them again. We repeated this wash step three times.

10. Finally, we performed the elution step to release the his-tagged protein (CEA N domain) bound to the beads. We adjusted the imidazole concentration sequentially to 100mM, 250mM, and 500mM to increase elution strength. We extracted the supernatant by centrifuging without any incubation time.

###### [Test 4: ‘Verifying the purified protein by Coomassie Brilliant Blue G-250 dye’]

---

[\*Coomassie Brilliant Blue G-250 dye\*]
The Coomassie Brilliant Blue G-250 dye assay is commonly used to detect proteins in a sample. Under acidic conditions, the dye interacts with proteins, causing a shift in color from reddish-brown to blue, which can be measured by absorbance at 595 nm. [8]

---

When you treated the purified sample with this stain and observed no color change, it indeed suggests that there is either a very low amount or an absence of purified protein in the sample. This conclusion indicates that the purification process did not proceed as expected or may have encountered issues, such as protein loss or insufficient purification.

---

###### [Learn 4: ‘Troubleshooting for the purification process, and optimization of protein purification protocol’]

The reasons for the improper purification were as follows:

 1. When preparing the cell lysate, we initially used 5 ml of lysis buffer to resuspend the cells after centrifuging 100 ml of cell culture. However, we divided this lysate into 2ml Eppendorf tubes, resulting in a significant dilution of the expressed protein. Therefore, we concluded that it would be more appropriate to resuspend 100ml of cultured cells with 2ml of lysis buffer in subsequent purification steps.

< 2. During the buffer preparation process, lysozyme and benzonase were accidentally added to both the washing buffer and elution buffer. Originally, lysozyme and benzonase were supposed to act only during the lysis step. Since it was uncertain how lysozyme and benzonase might have affected the purified protein during storage at 4°C after the purification process, it became **necessary to remake the buffers.**

 3. The step involving the addition of lysis buffer and subsequent centrifugation is the stage where cell lysate is created. In the protocol we referenced originally, it was stated to be done for 20-30 minutes. However, it's important to note that centrifugal force is proportional to the size and rpm (rotations per minute) of the centrifuge. Therefore, even at the same rpm, a larger centrifuge can exert more powerful separation. In our case, we used 2ml E-tubes and a smaller centrifuge, so **we adjusted the centrifugation time to 45 minutes for our procedure.**

< 4. During the binding process, we inadvertently applied excessive shaking in terms of both duration and intensity. We initially believed that vigorous shaking would lead to better mixing of the beads and protein, resulting in improved binding. Consequently, we applied strong shaking to ensure that the beads remained dispersed and did not settle. However, we soon realized that **this excessive shaking could cause even the bound protein to dissociate from the beads**. As a result, we decided to borrow a shaking device from a 4°C laboratory to conduct our experiments more effectively.

 5. For each Elution step, it is necessary to have an incubation period of about 10 minutes before centrifugation. Although it was not mentioned in the protocol we initially referred to, we decided to implement this step to improve protein purification. We believed that allowing sufficient time for imidazole in the elution buffer to bind to the beads and facilitate protein detachment was crucial for successful purification.

###### [Design 5: ‘Purifying the protein anew for EMSA’]

###### [Build 5: ‘Protein purification by Nickel bead’]

[optimized protein purification protocol]

We conducted protein purification using a protocol that had been optimized to suit our experimental needs.

For laboratories lacking column tubes specialized for protein purification, centrifuges compatible with 5ml tubes, or specialized protein purification kits, our protocol can serve as a valuable reference if they have access to a 2ml tube-compatible centrifuge and nickel or cobalt beads for purifying his-tagged proteins.

**Our method involves centrifugation followed by pipetting the supernatant, making protein purification feasible even in resource-constrained laboratory settings.**

We believe this information can be beneficial for individuals facing similar equipment and resource limitations.

For more detailed information, you can refer to the [experiments](https://2023.igem.wiki/kuas-seoul/experiments) section on the KUAS\_seoul wiki.

###### [Test 5: ‘Verifying the purified protein by Nanodrop’]

![ Figure 11. The Nanodrop analysis results for the protein nickel purification samples following cell culture at 30°C](https://static.igem.wiki/teams/4953/wiki/engineering1-11.png)

Most of the proteins that do not bind to the beads are extracted in the unbound step, resulting in the highest protein concentration. As we progress through the washing steps, such as wash 1, wash 2, and wash 3, proteins weakly bound to the beads should be washed away, leading to lower protein concentrations.

In the elution steps, elution 100 (imidazole concentration: 100mM), elution 250 (imidazole concentration: 250mM), and elution 500 (imidazole concentration: 500mM), protein concentrations should increase progressively.

In general, since the description aligns with the expected pattern, it can be inferred that purification has been to some extent successful. However, for a more precise assessment of the purification outcome, it is necessary to prepare samples with a consistent amount of purified protein and run SDS-PAGE gel electrophoresis.

https://static.igem.wiki/teams/4953/wiki/engineering1-12.png Figure 12. SDS-PAGE gel result

###### [Learn 5:'Troubleshooting']

After conducting the Nanodrop analysis, we determined the protein concentrations in the samples. Utilizing this information, we standardized the concentrations of the samples to ensure uniformity for an accurately controlled protein purification process.

However, upon further reflection regarding the overall low concentration, we realized that this was likely because we initially used only 100ml of cell culture when preparing the 2ml lysis buffer and conducting the purification. Consequently, to increase the protein concentration, we learned that it would be more effective to use 200ml of cell culture in the 2ml lysis buffer.

Additionally, the SDS-PAGE result shows that contamination was responsible for the observed outcomes. We opened the possibility that an E-tube from outside the lab, rather than sterilized E-tubes designated for the clean bench, was accidentally used to store the protein purification sample. This may have led to contamination in the sample, and it is possible that contamination occurred during the SDS sample preparation process as well.

###### [Design 6: ‘Purifying the protein anew for EMSA’]

###### [Build 6: ‘Protein purification by Cobalt bead’]

Generally, cobalt beads are noted to have higher specificity and lower affinity for His-tagged proteins compared to nickel beads. The active center of a **nickel-based resin** exhibits a more lenient spatial preference, allowing it to **interact with histidine residues located in positions other than the protein's polyhistidine tag. This results in increased yields but lower levels of purity.**

Hence, **we aimed to improve the purity of the CEA N domain protein by changing the type of beads used for protein purification. We maintained the same buffer composition and protocol throughout the process.**

###### [Test 6: ‘Verifying the purification of protein by Nanodrop’]

Most of the proteins that do not bind to the beads are extracted in the unbound step, resulting in the highest protein concentration. As we progress through the washing steps, such as wash 1, wash 2, and wash 3, proteins weakly bound to the beads should be washed away, leading to lower protein concentrations.

In the elution steps, elution 100 (imidazole concentration: 100mM), elution 250 (imidazole concentration: 250mM), and elution 500 (imidazole concentration: 500mM) were applied, in which the protein concentrations should also increase progressively.

In general, since the description aligns with the expected pattern, it can be inferred that purification has been to some extent successful. However, for a more precise assessment of the purification outcome, it is necessary to prepare samples with a consistent amount of purified protein and run SDS-PAGE gel electrophoresis. This part of the experiment is still ongoing.

![<Figure 13. The Nanodrop analysis results for the protein cobalt purification samples following cell culture at 37°C>](https://static.igem.wiki/teams/4953/wiki/engineering1-13.png)

###### [Learn 6: ‘CEA protein expression and purification optimization.’]

Through the DBTL process aimed at achieving the optimal expression of the CEA N domain protein, we have come to several key realizations:

 1. **Choosing a Different Strain** One crucial insight is the importance of going back to the beginning and selecting a different bacterial strain. We believe that transforming the desired plasmid into E. coli BL21, a strain specialized for protein expression, could yield larger quantities of the protein. In contrast to E. coli DH5α DE3, which exhibits lower protein expression levels, using nickel beads for purification instead of cobalt beads is more appropriate, as it allows for higher protein yields.

 2. **Optimizing Culture Temperature**: When using the BL21 strain, fine-tuning the culture temperature may be necessary to achieve the best results. **Temperature conditions around 30-37°C seem to be suitable**, but further optimization is likely needed.

**Utilizing the Entire Culture** To increase the concentration of purified protein, it's essential to utilize the entire 200ml cell culture during the protein purification process.

In the next cycle, we plan to build upon these lessons learned and conduct a completely new protein purification process. We will then proceed with EMSA experiments to determine whether the purified protein effectively binds to the aptamer, leveraging the knowledge gained during the learning process.

###### [Design 7: ‘EMSA: test of the binding of protein and aptamer.’]

Due to time constraints, we will continue with the EMSA experiment after uploading the iGEM page. For further information, please visit our notion!
[Notion](https://www.notion.so/kuas/KUAS-Korea-University-Association-of-Synthetic-Biology-1f5977b4e84c442dbce78f4ab62bf6bb?pvs=4)

💡“In 2023, Katalin Kariko, one of the Nobel laureates in Physiology or Medicine, encountered significant challenges in her career. After assuming the role of an assistant professor at the University of Pennsylvania, she applied for a research project on mRNA vaccines, only to face rejection, resulting in her departure from the position. However, undeterred by this setback, she persevered and continued her research as a non-regular researcher in a colleague's laboratory. Her journey eventually led to remarkable success, including the publication of influential work in Immunity in 2005 and, ultimately, her Nobel Prize win. In the field of synthetic biology, we can draw inspiration from Dr. Kariko's unwavering determination, never yielding when confronted with daunting challenges during the DBTL process. Currently, we are diligently working on formulating a new protocol tailored to our experiments, considering the insights from Dr. Kariko's journey. Operating within the constraints of a borrowed laboratory, we have encountered numerous limitations and hurdles due to restricted time and resources. Our commitment extends beyond the iGEM wiki submission deadline, as we are determined to persist with our experiments. We are resolute in our intention to present our findings at the grand jamboree." - Sujin Song

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Empirical evidence of research validity.{% endblock %}
{% block page\_content %}

# Introduction

---

The overall experimental process of our project is as shown in the figure below. On this page, we will provide detailed explanations for each of the steps.

![Figure 1. Experimental workflow](https://static.igem.wiki/teams/4953/wiki/engineering1-4.png)

## 1. H-Nuclear Magnetic Resonance (NMR) spectroscopy

**H-Nuclear Magnetic Resonance Spectroscopy (NMR)** is an analysis method that uses the spin properties and magnetic moments of atomic nuclei. This method is mainly used to analyze the properties of materials.

**The basic principles of H-NMR are as follows:**

 Certain atomic nuclei can have spin properties and magnetic moments. When these nuclei are exposed to a magnetic field, the energy levels of the rotating nuclei are separated. When energy levels are separated, the direction of the rotation axis changes as it transitions from a low energy level to a high energy level. At this time, the chemical surroundings affect the radio frequency range, and through this, it is possible to determine which atoms exist around the atom.

![“Fig.](https://static.igem.wiki/teams/4953/wiki/experiments17-3.png)

![Fig. 3. H-NMR spectroscopy analysis conducted by Seunghun Kim at Yonsei University Center of Research Facility(YCRF)](https://static.igem.wiki/teams/4953/wiki/experiments17-4.png)

![Fig. 4. H-NMR spectroscopy analysis conducted by Seunghun Kim at Yonsei University Center of Research Facility(YCRF)](https://static.igem.wiki/teams/4953/wiki/experiments17-5.png)

**When peaks related to the structure emerge through NMR spectroscopy, the integral value of each peak is obtained, and the integral value is compared with the H-NMR table [1] to analyze which peak it is for, and through this, the structure can be analyzed.**

![Fig. 5. H-NMR spectroscopy peak data table](https://static.igem.wiki/teams/4953/wiki/experiments17-6.png)

## 2. Protein-Aptamer Docking (PAD) experiment

To perform the PAD experiment, we first performed H-Nuclear Magnetic Resonance(NMR) spectroscopy to analyze the the structure of CEA protein and N56 aptamer. Furthermore, the structure of the protein-aptamer complex was also analyzed by H-NMR spectroscopy, the data was compared with the data before binding. That is, the H-NMR data for each protein aptamer before binding and the data for the binding complex were compared and analyzed. As a result, it was possible to analyze the binding pattern of the CEA and aptamer binding complex. Finally, the results of this analysis were visualized through a PAD experiment.

**The imaging results analyzing the binding pattern of CEA protein and N56 aptamer are as follows.**

Data deleted after the judging session

As we observed in PAD images, the bottom end of the aptamer is free to bind to the LFA sheet, and the N56 aptamer binds perpendicularly to the CEA.

To use aptamers in LFA, they must be bound nearly vertically, as shown in the image here.

**Why is it important for an aptamer to exhibit nearly perpendicular binding to a biomarker to be considered suitable for LFA?**

1.In any type of LFA sensor, the aptamer is fixed at one end. Chemically, the only location where the aptamer can be anchored arbitrarily while maintaining its function is at the end of the aptamer.

2.Various chemical repulsion forces, especially steric hindrance, occur very strongly near parts that have already been chemically bonded, that is, fixed. This is a very self-evident fact, and in one of the many papers reporting this, a Paper published in ACS Journal stated: "Due to steric hindrance, aptamers immobilized close to the surface may not be able to fold into the three-dimensional structure necessary for target recognition.” [2]

3.Therefore, It is very unlikely that a relatively large biomarker will be attached to the side of the aptamer immobilized on the LFA sheet by overcoming the enormous steric hindrance. (Of course, if the aptamer is extremely long and straight, the biomarker can bind to the side, but such aptamer is structurally very unstable and is not suitable for use as a probe.)

4.In other words, an aptamer suitable for LFA must bind a biomarker to one loop or end excluding the fixed end. **When this is satisfied, this document expresses this as “binding almost vertically (or perpendicularly).”**

**In conclusion, the nearly vertical binding of the CEA protein and N56 aptamer was visualized through experiments.
This experimental discovery demonstrates for the first time the potential application of an LFA assay-based CEA protein sensor using N56 aptamer, and these images are being released for the first time in iGEM.**

## 3. Cell-Free Expression of CEA N domain

### Cell-Free Expression

Cell-free protein synthesis was carried out using previously designed template DNA to minimize contamination that may occur in biological experiments and to quickly obtain proteins without using cells. A cell-free protein synthesis experiment was conducted using the ‘NEBExpress cell-free protein synthesis kit’, and 10 reactions were performed in the first step. Through this experiment, it was confirmed that the CEA N domain protein was expressed, but a sufficient amount of protein was not obtained to be used in the EMSA experiment. To secure additional CEA N domain protein, E. coli was transformed twice to obtain a DNA template. Afterward, a second cell-free protein synthesis was performed using the additionally obtained DNA template.

The preparations, protocol[3], and results of the experiment are as follows.

Preparations (Cell-Free Protein Synthesis)

```
NEBExpress S30 Synthesis Extract

Protein Synthesis Buffer (2X)

T7 RNA Polymerase (Supplied in 50 mM Tris-HCl, 100 mM NaCl, 20 mM β-ME, 1 mM EDTA, 50% glycerol, 0.1% Triton****™**** X-100, pH 7.9)

RNase Inhibitor, Murine (Supplied in 20 mM HEPES-KOH, 50 mM KCl, 8 mM DTT, 50% glycerol, pH 7.6)

NEBExpress Control DHFR-His Plasmid (Encoding E.coli dihydrofolate reductase)

Incubator

Pipette

Microcentrifuge tube
```

Cell-Free Expression protocol

```
1.Thaw all components (NEBExpress S30 Synthesis Extract, protein synthesis buffer (2X), T7 RNA polymerase, RNase inhibitor, Murine, and NEBExpress control DHFR-His plasmid) on ice.

2.Gently vortex the NEBExpress S30 Synthesis Extract and protein synthesis buffer to mix.

3.Combine reagents in a 1.5ml microcentrifuge tube on ice as follows:

| Components | Negative control | Positive control | Sample |
| --- | --- | --- | --- |
| NEBExpress S30 Synthesis Extract | 12 μl | 12 μl | 12 μl |
| Protein synthesis buffer (2X) | 25 μl | 25 μl | 25 μl |
| T7 RNA polymerase | 1 μl | 1 μl | 1 μl |
| RNase inhibitor, Murine | 1 μl | 1 μl | 1 μl |
| NEBExpress control DHFR-His plasmid (125 ng/μl) | - | 2 μl | - |
| Plasmid template (>100 ng/μl) | - | - | 250 ng |
| Water | 11 μl | 9 μl | to 50 μl |

4.Incubate reactions at 37℃, with vigorous shaking, for 2-4 hours.
5.Analyze by method of choice or freeze at -20℃ for later use.

  file[\kuas-seoul\updatefiles\NEBExpress_CFE_manual.pdf]
```

Preparations (Transformation)

```
DH10B (Plasmid cloning strain)

DNA template

Lysogeny broth (LB)

Shaker

Centrifuge

LB plate

Glass beads
```

Transformation protocol

```
[Plasmid Cloning]

1. Add the 80 ng/µL of GOI (gene of interest) in the aliquoted competent cell.
2. Thaw on ice for 20 minutes.
3. Apply heat shock at 42˚C for 45 seconds.
4. Thaw on ice for 2 minutes.
5. Add 800 µL of warm LB on each tube.
6. Vigorously shake samples at 200 rpm, 37˚C at the shaker for 1 hour.
7. Centrifuge the tubes at 13,000 rpm for 1 min at RT.
8. Re-suspend 100 µL of warm LB.
9. Apply 100 µL of the solution on each LB plate (including antibiotics) with glass beads/spreader.
```

Result
![“Fig.](https://static.igem.wiki/teams/4953/wiki/11-dna-transformation.jpg)
![“Fig.](https://static.igem.wiki/teams/4953/wiki/12-cea-n-domain-transformation-success.jpg)

```
We were able to successfully transform the CEA N domain gene sequence using the DH10B strain.
```

## SDS-PAGE

SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a technique for classifying proteins by molecular weight. SDS-PAGE eliminates the effects of protein structure and charge and simply classifies proteins based on molecular size. SDS is an anionic detergent that denatures proteins and destroys their structures. After SDS treatment, the protein is uniformly coated with a negative charge proportional to its molecular weight, and this coating neutralizes the intrinsic charge of the protein, making it simple to classify by size during electrophoresis. Polyacrylamide gels act as a medium by which proteins travel when exposed to an electric field.

SDS-PAGE was performed to check whether the protein was properly expressed after expressing the CEA N domain with the cell-free expression system. According to the reference paper[4], purified CEA N domain represents about 13 kDa band. In the case of KUAS' study, template DNA was designed to express 6x His tag together in the CEA N domain, so it could be expected that a protein band of approximately 16 kDa would appear.

The preparations, protocol[5], and results of the experiment are as follows.

Preparations (SDS-PAGE)

```
 PageRuler™ Unstained Low Range Protein Ladder

 2 M Tris-HCl (pH 8.8)

 1 M Tris-HCl (pH 6.8)

 10% SDS (w/v)

 50% glycerol (v/v)

 1% bromophenol blue (w/v)

 1. Solution A (Acrylamide stock solution)

  30% (w/v) acrylamide

  0.8% (w/v) bis-acrylamide

 2. Solution B (4x separating gel buffer)

  2 M Tris-HCl (pH 8.8)

 10% SDS

 H₂O

 3. Solution C (4x stacking gel buffer)

  1 M Tris-HCl (pH 6.8)

  10% SDS

  H₂O

 4. 10% ammonium persulfate
 5. Electrophoresis buffer

  Tris

  Glycine

  SDS

  H₂O

6. 5x sample buffer

  1 M Tris-HCl (pH 6.8)

  50% glycerol

  10% SDS

  2-mercaptoethanol

  1% bromophenol blue

  H₂O

  Solution A

  Solution B

  Solution C

  H₂O

  10% ammonium persulfate

  TEMED
```

SDS-PAGE protocol

```
 1. Combine NEBExpress Cell-free Protein Synthesis System reaction with 5x sample buffer. Also, prepare a negative control sample.
 2. Incubate at 100℃ for 5 minutes.
 3. Load 5 μl of the standard protein marker into the first lane.
 4. After a quick microcentrifuge spin, load 5 μl of samples directly onto the 15% gel. To ensure uniform mobility, load an equal volume of SDS-PAGE Blue loading buffer into any unused wells.
 5. Run the gel at 80-120V until the protein is completely separated by molecular weight.
 6. Stain with Coomassie Blue stain buffer overnight.
 7. Destain with destain buffer for overnight.
 8. Analyze the location and thickness of the band appearing in the gel.
```

Result

```
![“Fig.](https://static.igem.wiki/teams/4953/wiki/experiments17-3.jpg)

In the left gel, the SDS-PAGE results showed, in order, marker ladder, positive control, negative control, samples 2, 3, 4, 5, and 6. In the right gel, the SDS-PAGE results showed, in order, marker ladder, negative control, positive control, samples 1, 2, 3, 4, 5, and 6.

It was confirmed that the CEA N domain protein was not well expressed in the cell-free expression experiment.

![“Fig.](https://static.igem.wiki/teams/4953/wiki/experiments17-4.jpg)

In the upper gel, the SDS-PAGE results showed, in order, marker ladder, negative control, and positive control, samples 6, 7, 8, 9, 10, and 11. In the lower gel, the SDS-PAGE results showed, in order, marker ladder, negative control, positive control, samples 1, 2, 3, 4, and 5.

Through this result, it was confirmed that the CEA N domain protein was well expressed in the cell-free expression experiment, but not in sufficient quantities for use in the EMSA experiment.
```

---

### Protein purification

The CEA N domain protein was purified for EMSA with N56 DNA aptamer. For rapid screening and purification of 6x His-tagged proteins from small-scale expression cultures, the protein was purified using a Ni-NTA spin column.

The preparations, protocol[6], and results of the experiment are as follows.

Preparations (Protein purification)

```
        (pH 8.0)

        50mM NaH2PO4

        300mM NaCl

        10mM imidazole

         (pH 8.0)

        50mM NaH2PO4

        300mM NaCl

        20mM imidazole

         (pH 8.0)

        50mM NaH2PO4

        300mM NaCl

        500mM imidazole

        Benzonase endonuclease 25 U/µL

        Lysozyme stock solution 10 mg/mL in water

        Ni-NTA spin columns
```

Protein purification protocol

```
1. Incubate columns for protein purification on ice for 15-30 minutes
2. Centrifuge protein sample at 12,000 x g for 15-30 min at 4℃. Collect supernatant.
3. Equilibrate the Ni-NTA spin column with 600 µL buffer NPI-10. Centrifuge for 2 min at 890 x g (approx. 2900 rpm).
4. Load up to 600 µL of the protein sample containing the 6x His-tagged protein onto the pre-equilibrated Ni-NTA spin column. Centrifuge for 5 min at 270 x g (approx. 1600 rpm), and collect the flow-through.
5. Wash the Ni-NTA spin column twice with 600 µL buffer NPI-20. Centrifuge for 2 min at 890 x g (approx. 2900 rpm).
6. Elute the protein twice with 300 µL buffer NPI-500. Centrifuge for 2 min at 890 x g (approx. 2900 rpm), and collect the eluate.
7. To confirm whether the protein is well purified, make a protein sample using part of the purified protein, perform SDS-PAGE, and check the results.

  file[\kuas-seoul\updatefiles\EN-Ni-NTA-Spin-Kit-Handbook.pdf]
```

Result

```
          ![Fig. 16. The purification results of the CEA N domain”](https://static.igem.wiki/teams/4953/wiki/cfe-purification.jpg)

The protein produced via cell-free expression was purified and its identity was verified using SDS-PAGE analysis. However, the analysis revealed that there was no detectable protein band at the expected size of 13 kDa. For a more comprehensive description of the DBTL process related to this issue, please refer to the engineering section.
```

## CEA N domain expression in cells

Although the CEA N domain was expressed with cell-free expression, a sufficient amount of protein was not obtained to be used in EMSA. This suggests the possibility that by optimizing the experimental conditions of CFE, a sufficient amount of protein can be obtained and synthetic biology techniques such as CFE can be used to confirm the binding affinity between aptamers and biomarkers of other disease, to proceed as quickly as possible with EMSA, which checks the binding of N56 aptamer and CEA N domain, it was intended to express the CEA N domain in the cell.

Therefore, to proceed as quickly as possible with EMSA, which checks the binding of N56 aptamer and CEA N domain, it was intended to express the CEA N domain in the cell.

We conducted protein purification using Ni-NTA nickel beads and Ni-NTA cobalt beads due to a shortage of spin columns. Additionally, as native proteins needed to be purified, we used NaH2PO4 instead of tris for protein purification. The optimization process for CEA N domain protein purification (DBTL process) can be found in the [engineering](https://2023.igem.wiki/kuas-seoul/engineering) section.

The preparations, protocol, and results of the experiment are as follows.

Preparations (CEA N domain expression in cells)

CEA N domain protein expression

```
DH10B (negative control)

DH5α DE3 (Sample)

DNA template

Lysogeny broth (LB)

Shaker

Centrifuge

LB plate

Glass beads
```

Protein purification

```
 (pH 8.0)

50 mM NaH2PO4

300 mM NaCl

10 mM Imidazole

 (pH 8.0)

50 mM NaH2PO4

300 mM NaCl

20 mM Imidazole

 (pH 8.0)

50 mM NaH2PO4

300 mM NaCl

100 mM Imidazole

 (pH 8.0)

50 mM NaH2PO4

300 mM NaCl

250 mM Imidazole

 (pH 8.0)

50 mM NaH2PO4

300 mM NaCl

500 mM Imidazole

Lysozyme

Benzonase endonuclease

2 mL tube

1.5 mL tube

Ni-NTA agarose bead

Cobalt resin

Centrifuge
```

Protein synthesis protocol

CEA N domain protein expression
**[Plasmid cloning & CEA N domain expression**

```
1. Add the 80 ng/µL of GOI (gene of interest) in the aliquoted competent cell.
2. Thaw on ice for 20 minutes.
3. Apply heat shock at 42˚C for 45 seconds.
4. Thaw on ice for 2 minutes.
5. Add 800 µL of warm LB on each tube.
6. Vigorously shake samples at 200 rpm, 37˚C at the shaker for 1 hour.
7. Centrifuge the tubes at 13,000 rpm for 1 min at RT.
8. Re-suspend 200 µL of warm LB.
9. Apply 200 µL of the solution on each LB plate (including antibiotics) with glass beads/spreader.
10. After completing step 9, allow one hour to elapse, and then commence measuring optical density (OD) at 15-minute intervals.
11. Incubate each sample for 4 hours at 37˚C, 30˚C, and 16˚C with continuous agitation at 150 rpm. Additionally, include 200 µL of a 1000x IPTG solution in the incubation process.
12. Centrifuge the cells at 4˚C and 3000 rpm for 35 minutes.
13. Discard the flowthrough and retain only the supernatant.
14. To verify the successful expression of the protein, an expression test will be performed.
```

The chosen strain for protein expression: E. coil (DH5alpha\_DE3)

```
1. Versatility: E. coli is a commonly used host organism for protein expression in research and biotechnology due to its well-characterized genetics and the availability of various expression vectors.

2. Rapid Growth: E. coli has a short generation time, allowing for quick production of protein.

3. Strong Promoters: The DH5alpha_DE3 strain contains a T7 RNA polymerase gene under the control of the lacUV5 promoter, which can be induced by adding IPTG (Isopropyl β-D-1-thiogalactopyranoside). This system enables tight control of protein expression.

4. High Yield: E. coli can produce a high yield of recombinant protein when optimized for the specific protein of interest.
```

Protein purification

```
[Ni-NTA agarose bead]

[Lysis]>

1. Thaw the cell samples on ice for 15 minutes.
2. Add 2 mL of lysis buffer to the downed cell and resuspend it.
3. Add 1.4 mL of lysozyme(10 mg/mL) and 1.2µL (250 U/µL) of benzonase endonuclease to the cell sample.
4. Incubate samples on ice for 30 minutes.
5. Centrifuge the samples at 10,000 x g for 45 minutes at 4°C.
6. Take 1.6 mL of lysate by the ratio of Ni-NTA agarose bead.
7. Add 400 µL of Ni-NTA agarose bead to a 2 mL tube and centrifuge at 3,000 rpm for 5 minutes at 4°C.
8. Discard the supernatant, preservation solution.
9. Put 1.5 mL of lysis buffer into a 2 mL tube containing Ni-NTA agarose bead and centrifuge at 3,000 rpm for 5 minutes at 4°C for washing.
10. Discard the supernatant.
11. Repeat step 9.
12. Repeat step 10.
13. Put lysate taken in step 6 into a 2 mL tube containing Ni-NTA agarose bead.

        **[Binding]**

        1. After agitation, shake at 4°C for 1 hour at an appropriate intensity.
        2. Centrifuge the samples at 800 x g for 10 minutes at 4°C.
        3. Remove the supernatant containing unbound protein for subsequent SDS-PAGE.

        **[Washing]**

        1. Add 1.6 mL of wash buffer to the sample, agitate, and centrifuge at 800 x g for 5 minutes at 4°C.
        2. Remove the supernatant, store it separately, and repeat step 17 (1st washing).
        3. Remove the supernatant, store it separately, and repeat step 17 (2nd washing).
        4. Remove the supernatant as much as possible, and store it separately (3rd washing).

        **[Elution]**

        1. Add 500 µL of 100 mM imidazole elution buffer to the sample.
        2. Incubate samples on ice for 10 minutes.
        3. Centrifuge the samples at 500 x g for 10 minutes at 4°C.
        4. Take the supernatant and add 500 µL of 250 mM imidazole elution buffer to the sample.
        5. Repeat steps 22 and 23.
        6. Take the supernatant and add 500 µL of 500 mM imidazole elution buffer to the sample.
        7. Repeat steps 22 and 23.
        8. Take the supernatant.

        **[Cobalt resin]**

        **[Lysis]**

        1. Thaw the cell samples on ice for 15 minutes.
        2. Add 2 mL of lysis buffer to the downed cell and resuspend it.
        3. Add 1.4 mL of lysozyme(10 mg/mL) and 1.2µL (250 U/µL) of benzonase endonuclease to the cell sample.
        4. Incubate samples on ice for 30 minutes.
        5. Centrifuge the samples at 10,000 x g for 45 minutes at 4°C.
        6. Take 1.6 mL of lysate by the ratio of cobalt resin.
        7. Add 400 µL of cobalt resin to a 2 mL tube and centrifuge at 3,000 rpm for 5 minutes at 4°C.
        8. Discard the supernatant, preservation solution.
        9. Put 1.5 mL of lysis buffer into a 2 mL tube containing cobalt resin and centrifuge at 3,000 rpm for 5 minutes at 4°C for washing.
        10. Discard the supernatant.
        11. Repeat step 10.
        12. Repeat step 9.
        13. Put lysate taken in step 6 into a 2 mL tube containing cobalt resin.

        **[Binding]**

        1. After agitation, shake at 4°C for 1 hour at an appropriate intensity.
        2. Centrifuge the samples at 800 x g for 10 minutes at 4°C.
        3. Remove the supernatant containing unbound protein for subsequent SDS-PAGE.

        **[Washing]**

        1. Add 1.6 mL of wash buffer to the sample, agitate, and centrifuge at 800 x g for 5 minutes at 4°C.
        2. Remove the supernatant, store it separately, and repeat step 17 (1st washing).
        3. Remove the supernatant, store it separately, and repeat step 17 (2nd washing).
        4. Remove the supernatant as much as possible, and store it separately (3rd washing).

        **[Elution]**

        1. Add 500 µL of 100 mM imidazole elution buffer to the sample.
        2. Incubate samples on ice for 10 minutes.
        3. Centrifuge the samples at 500 x g for 10 minutes at 4°C.
        4. Take the supernatant and add 500 µL of 250 mM imidazole elution buffer to the sample.
        5. Repeat steps 22 and 23.
        6. Take the supernatant and add 500 µL of 500 mM imidazole elution buffer to the sample.
        7. Repeat steps 22 and 23.
        8. Take the supernatant.
```

SDS-PAGE

```
1. Combine protein sample with 5x sample buffer. Also, prepare a control sample.
2. Incubate at 100℃ for 5 minutes.
3. Load 5 μl of the standard protein marker into the first lane.
4. After a quick microcentrifuge spin, load 5 μl of samples directly onto the 15% gel. To ensure uniform mobility, load an equal volume of SDS-PAGE Blue loading buffer into any unused wells.
5. Run the gel at 80-120V until the protein is completely separated by molecular weight.
6. Stain with Coomassie Blue stain buffer overnight.
7. Destain with destain buffer for overnight.
8. Analyze the location and thickness of the band appearing in the gel.
```

Results

Protein purification
![“Fig.](https://static.igem.wiki/teams/4953/wiki/experiments17-1.png)

Most of the proteins that do not bind to the beads are extracted in the unbound step, resulting in the highest protein concentration. As we progress through the washing steps, such as wash 1, wash 2, and wash 3, proteins weakly bound to the beads should be washed away, leading to lower protein concentrations.

In the elution steps, elution 100 (imidazole concentration: 100 mM), elution 250 (imidazole concentration: 250 mM), and elution 500 (imidazole concentration: 500 mM), protein concentrations should increase progressively.

## 5. EMSA(Electrophoretic Mobility-Shift Assay)

EMSA is an experimental method that can check the degree of binding between genes and proteins. The main principle is to label the DNA probe with radiation isotopes or non-radiation isotopes such as fluorescein to react with proteins at room temperature to determine the degree of band shift in electrophoresis to confirm the binding between proteins and genes.

EMSA was conducted to qualitatively check whether the N56 aptamer is well bound to the CEA N domain and to determine whether the N56 aptamer is appropriate for use in CEApture, LFA-type sensor.

While we were unable to conduct the EMSA experiment before the Wiki freezing, KUAS continues to progress with the experiments. We will endeavor to include any relevant information about the EMSA experiment results in a future presentation video, if possible.

The preparations, protocol[7], and results of the experiment are as follows.

Preparations(EMSA)

```
**EMSA running buffer (0.5x TBE running buffer)**

10x TBE buffer

Tris base

Boric acid

0.5 M EDTA (pH 8.0)

dH₂O

dH₂O

**Non-denature PAGE gel (6%)**

40% acrylamide

2% bis-acrylamide

10x TBE buffer

TEMED

10% ammonium persulfate

dH₂O

**2x gel shift reaction buffer**

50% glycerol

1 M HEPES (pH 7.9)

1 M Tris-HCl (pH 8.0)

0.5 M EDTA (pH 8.0)

100 mM DTT

dH₂O

BSA (1 µg/µL)

Poly (dI-dC) (0.5 µg/µL)

FAM-labeled N56 DNA aptamer

CEA N domain protein
```

EMSA protocol

```
1. Mix 12 μL of 2x reaction buffer, 3 μL of BSA, 2 μL of poly (dI-dC), 3 μL of CEA N domain protein, and 3 μL of dH₂O, then incubate at room temperature or on ice for 30 minutes.
2. Add 1 μL of FAM-labeled N56 DNA aptamer.
3. Keep at room temperature for 20 minutes.
4. Load the gel and run at 200 V for 1 hour to 1 hour and 30 minutes. Use the DNA loading buffer in lane 1 as an indicator of the free probe.
5. Stop the gel when the dye runs at 3 cm to the bottom.
6. Dry the gel and expose the dried gel to X-ray film at -70˚C overnight.
7. Develop the film.
```

### Reference

```
[1] Gottlieb, H. E., Kotlyar, V., & Nudelman, A. (1997). NMR chemical shifts of common laboratory solvents as trace impurities. Journal of organic chemistry, 62(21), 7512-7515.

[2]  Johanna-Gabriela Walter, Öznur Kökpinar, Karl Friehs, Frank Stahl, and Thomas Scheper Analytical Chemistry 2008 80 (19), 7372-7378 , DOI: 10.1021/ac801081v

[3] NEW ENGLAND BioLabs, NEBExpress Cell-Free E. Coli Protein Synthesis System. NEW ENGLAND BioLabs.

[4] A. Krop-Watorek, S. Oikawa, Y. Oyama, and H. Nakazato, “Oligomerization of N-terminal domain of carcinoembryonic antigen (CEA) expressed inEscherichia Coli,” Biochemical and Biophysical Research Communications*, vol. 242, no. 1, pp. 79–83, 1998. doi:10.1006/bbrc.1997.7920

[5] D. M. Bollag, M. D. Rozycki, and S. J. Edelstein, Protein Methods. New York: Wiley-Liss, 1996.

[6] QIAGEN, Ni-NTA Spin Kit Handbook, 2nd ed. QIAGEN, 2008. https://www.qiagen.com/us/resources/resourcedetail?id=3fc8c76d-6d21-4887-9bf8-f35f78fcc2f2⟨=en

[7] J. Y. MD, Gel Shift / EMSA Protocol. Pennington Biomedical Research Center.
```

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Main results for 'CEApture'{% endblock %}
{% block page\_content %}

### Results

###### **PART 1** | in silico Design & Estimation

| N54 | N56 | N57 | N59 | N65 | N71 | cApt | Docking score | -219.07 | -226.66 | -218.33 | -226.18 | -219.93 | -217.00 | -221.87 | Confidence score | 0.7992 | 0.8225 | 0.7968 | 0.8211 | 0.8020 | 0.7925 | 0.8081 |

As confronted with practical constraints, we utilized in-silico program, **HDock**, to confirm that N56 is the best aptamer candidate for CEA. The results of the docking score demonstrated a significant level of affinity among the six aptamer candidates, making them suitable for utilization in LFA. The binding score was calculated based on aptamer and protein sequences.

---

###### **PART 2** | Binding pattern analysis and visualization

Data deleted after the judging session

These images are the results of the Protein-Aptamer Docking(PAD) experiment. To use aptamers in LFA, they must be bound vertically with protein due to steric hindrance [1]. From PAD images, we confirmed that N56 aptamer binds perpendicularly to the CEA.

As a result, the visualization of the nearly perpendicular coupling between the CEA-N56 pair demonstrates its suitability for LFA sensors, which has not been reported in previous papers. We report this for the first time in iGEM.

###### **PART 3** | CFE & EMSA

1.**CFE** (**C**ell-**F**ree-**E**xpression)

One of our goals in synthetic biology is the production of proteins through a Cell-Free Expression system (CFE). CFE is anticipated to emerge as a potent tool in the future of synthetic biology. Therefore, we executed a Design-Build-Test-Learn (DBTL) cycle to refine the environmental conditions of the protein expression process.
During the initial CFE cycle, we encountered challenges in the overexpression of CEA proteins. Subsequently, we conducted troubleshooting to solve the underlying issues in the experimental conditions and proceeded with a second cycle of CFE.

2.**EMSA**(**E**lectrophoretic **M**obility **S**hift **A**ssay)

The EMSA serves as a powerful tool for confirming the in vitro binding between specific nucleic acids and proteins. In our study, we aimed to demonstrate the interaction between CEA and the N56 aptamer using this technique. The observation of band-shift in the mobility of the FAM-tagged aptamer, when compared to controls, provides evidence of the binding event.

The Electrophoretic Mobility Shift Assay (EMSA) is a valuable tool for confirming in vitro interactions between specific nucleic acids and proteins. Our study aimed to use this technique to demonstrate the interaction between CEA and the N56 aptamer. This demonstration is usually achieved by observing a band shift in the mobility of the FAM-tagged aptamer when compared to control samples.

Regrettably, due to constraints, including tight deadlines, we were unable to carry out the EMSA as planned. Nevertheless, KUAS remains committed to advancing our experiments. We are determined to incorporate any relevant findings from the EMSA experiment into an upcoming presentation video as soon as circumstances permit.

Despite the absence of an EMSA experiment, we are confident in the binding interaction between the N56 aptamer and CEA. This confidence is grounded in prior research and the results from our modeling and PAD experiments.

### Discussion

###### **PART 1** | Advantages & Significance

Here are the advantages and significance of this research.

Firstly, We designed CEA N domain DNA templates(parts) for in vitro expression(CFE). There are only a few DNA template parts for CFE, it would be a great milestone for future iGEM projects.

Secondly, uploading aptamers to the parts registry along with corresponding documents contributes to the accessibility and dissemination of iGEM projects.

Additionally, proposing a new paper-based diagnostic device signifies progress in **point-of-care** technology, improving the practicality of medical diagnostics. The revealed analysis of the CEA-N56 binding complex provides insights into potential biosensor applications for the cervical cancer biomarker CEA. The observed near-vertical binding pattern highlights the potential for sensor development, going beyond the identification of specific aptamers.

###### **PART 2** | Further Research Suggestions

In our future research, we will focus on advancing the "CEApture". As we proved the aptamer N56 and the biomarker CEA are suitable for quick diagnosis called **Lateral Flow assays(LFA)**, quantifying how well these elements bind together is needed. The dissociation constant, Kd, might be measured by a powerful method called **Confocal Laser Scanning Microscopy(CLSM)**.

To make it commercialized in the real world, our team plans to design detailed LFA sensors, especially focusing on color-changing methods/two-line indications on the pads. The fact that we've already proven gives us enough confidence that "CEApture" will work well in real life. We hope that it will help more people accept and use LFA technology for cancer diagnosis in their lives.

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In conclusion, our experimental findings proved the clinical viability of the CEA protein and N56 aptamer pairing for application in an LFA sensor. This establishes a scientific foundation for the development of the LFA sensor-based pad that can be used as cervical cancer diagnostic device, termed 'CEApture.'
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### Reference

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[1] J.-G. Walter, Ö. Kökpinar, K. Friehs, F. Stahl, and T. Scheper, “Systematic Investigation of Optimal Aptamer Immobilization for Protein−Microarray Applications,” Analytical Chemistry, vol. 80, no. 19, pp. 7372–7378, Aug. 2008, doi: https://doi.org/10.1021/ac801081v.
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