Notebook

 📖 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.

 

        

          

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

📖 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 
          
      
 
    

📖 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 
 
 
 
            

📖 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 
          
      
 
    

📖 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
 
 
 
            

📖 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 
 
 
 
   

📖 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
 
 
 
            

📖 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 
 
 
 
            

📖 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.

  

    
  
  


  

         

        

          

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

 📖 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 
 
 
 
            

      
📖 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.


  

    







    

        

          

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

📖 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.



 

        

          

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

📖 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. 


 

        

          

            
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
          
            

📖 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
 
 
 
            

      
📖 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!




    

        

          

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

📖 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.




 

        

          

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

📖 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 
 
 
 
            

📖 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