Overview

We valued our involvement in Human Practices activities in the project from a larger perspective than just within the laboratory, expanding directly to our consumers and possible target markets.

To this end, we were conscious of the following two points.
(1) Engage with various stakeholders and exchange opinions through two-way communication.
(2) Reflect on human practice discussions and ensure that they are reflected in the project.

To make the project better, we needed real voices. By actually talking to the stakeholders, we achieved the following outcomes: HP’s activities had the following impact on us: product decisions, thinking towards implementation, technical support, and sharing and discussing the project.

The main results include the following
・By talking to a B. subtilis Natto expert, we were able to provide plasmids and solve technical problems.
・Visiting a senior citizen’s home to find out the best product to use.
・By visiting the government’s food guidance department, we were able to think deeply about the safety of healthy food products.

We used the Human Practice Cycle to integrate HP activities in a thoughtful way that allowed us to identify, dialogue, reflect, and act on issues.
As a result, we were able to make VitaliNK a better place for the world.

Human Practice Cycle

To integrate the project into something good for society, it is important to reflect on HP’s activities in the project. We used Gibb’s Reflection Cycle, produced for learning from experience. This method, published by Graham Gibbs in. Learning by DoingLearning by Doing: A Guide to Teaching and Learning Methods, was optimized by the iGEM team greatbay-scie of 2023
We used them as a reference to produce a CYCLE that we further optimized for our project.
This turned out to be a very useful tool for how to integrate discussions with stakeholders.

Impetus and Defining Purpose

This step presents the Impetus to do human practice and brush up on project issues.

Where are we stuck in our project?
What feedback is missing in our project?
Who are the stakeholders that our project doesn’t consider yet?

After clearly defining these issues, we plan how to promote understanding and to whom.

Two Way Communication

Two-way communication is where we provide the stakeholders with the project details and help them understand our project.
Understanding the project would ensure that our concerns and suggestions would be understood and that we would receive consistent feedback.

Analysis

Based on the feedback we received from the two-way communication, we analyzed and reflected on our project.

What are the problems we are facing?
What are the circumstances behind the feedback we received?
What were the important points in the feedback we received?

The purpose of this section is to determine the status of our project based on what we learnt in the two-way communication.

Implementation

The purpose of this section is to apply and integrate what we have evaluated in the analysis to our project, and to identify actions we can take to improve on the problems and objectives we have identified in the “impetus and defining purpose” step. We will also detail how we implemented and applied them, and what impact they had on the overall project.

Project Decision

Awareness of Thrombosis

Thrombosis accounts for one in four deaths worldwide(1). It is a general term for diseases caused by blood clots obstructing blood vessels, including myocardial infarction, stroke, pulmonary embolism, and cardiogenic stroke.

This is a significant issue in Japan, which is experiencing an aging society. Among the G7 countries, Japan has the highest percentage of elderly people within its total population. According to data from 2022, while the rate of individuals aged 65 and older in the U.S. is 17%, it is estimated to be around 30% in Japan(2).

Statistics show that 66% of stroke deaths occur in individuals aged 70 and older(3). Age is a primary risk factor for Thrombosis, and with the aging population, the burden of cardiovascular diseases is increasing. The second leading cause of death in Japan is heart disease, including myocardial infarction, while cerebrovascular disorders rank third. It is believed that approximately 30% of deaths in Japan are due to thrombosis.

There are various types of thrombosis, but here we will focus on two particularly severe conditions.

Strokes

“Stroke” is a general term for diseases caused by blood clots’ obstruction of blood vessels in the brain, leading to insufficient blood supply to brain cells and subsequent cell necrosis. According to WHO data from 2021, strokes ranked as the third leading cause of death globally(4), with approximately 6.6 million deaths predicted in 2022(3). In Japan, cerebrovascular diseases accounted for 6.6% of deaths in 2023, with over 10,000 fatalities(5).

In caregiving settings, cerebrovascular disorders pose a significant challenge. In Japan, 16.1% of patients requiring care cite cerebrovascular disorders as the primary cause of their condition(6).

Acute Myocardial Infarction

Acute myocardial infarction occurs when blood vessels are blocked by plaque or blood clots, preventing blood flow and resulting in insufficient delivery of nutrients and oxygen to the heart muscle, leading to its necrosis. In 2023, over 30,000 deaths due to acute myocardial infarction were confirmed in Japan(5). Additionally, the economic losses associated with myocardial infarction are substantial. The economic burden of ischemic heart diseases, including myocardial infarction, was estimated at 1.619 trillion yen in Japan in 2017(7).

Additionally, between one-third and one-half of hospitalized patients with acute myocardial infarction are elderly individuals aged 70 and older(8). 80% of deaths from myocardial infarction occur in those aged 65 and older, similar to strokes, indicating that the majority of patients affected by these conditions are elderly(8).

In this way, thrombosis represents a critically significant issue in Japan’s rapidly aging society, as well as a major global challenge that must be addressed.

Prof. Osamu Maruyama

To better understand the current situation caused by thrombosis, we consulted with Prof. Osamu Maruyama, who works in the Department of Biomedical Engineering for Health at the National Institute of Advanced Industrial Science and Technology and is involved in the development of artificial hearts and research on the mechanisms of blood clotting.

Impetus and Defining Purposes

We had been conducting an online literature review on thrombosis but were limited in our own knowledge of the uses and side effects of the currently attached therapeutic agents, and were unable to deepen our ideas on effective ways to solve thrombosis.
Therefore, we contacted Prof.Osamu Maruyama, who conducts basic research on blood coagulation, to gain real information, experience, and new knowledge about the medical field and current treatment methods.

Two-Way Communication

It was pointed out to us that we needed to clarify our problem setting, as “we want to solve thrombosis” alone is too unclear a goal. Therefore, we focused one nurturing a mutual conversation, as we explained our project to them and asking for their advice which we later implemented in elements of our project.

He also suggested that our product should not be used in the medical field after the disease has developed, but rather should be promoted as a project aimed at prevention. In addition to the importance of not becoming ill in the first place, the current preventive measures against thrombosis, such as ischemic heart disease, have no realistic solution other than measures through lifestyle modification, such as salt reduction and exercise, in order to lower blood pressure.

We learned that current treatment methods mainly use anticoagulants and antithrombotic drugs for severely ill patients. We learned that anticoagulants are used only in critically ill patients who have a lot of artifacts in their bodies, such as dialysis.

Analysis

When it was pointed out that the problem setting was not clear, we realized that although we had a goal of “solving thrombosis,” we had not decided which specific segment of the population we would target. We felt that it was necessary to clarify whether we were supporting seriously ill patients, targeting those who are on the verge of developing the disease, or whether we were aiming for healthy people to use the product daily.
We also realized that the only preventive measures we can take daily against heart disease and other blood diseases are to reduce salt intake, exercise more, and improve our lifestyles. Thus, we thought there could be a way to intervene with this situation and make day-to-day prevention through food a common practice.

Implementation

▶We decided to create our project based on and targeted preventive foods.=
Through this activity, we became convinced that if the most effective way to prevent thrombosis is to be careful about one’s lifestyle, then we need a product that is easy to use and effective against thrombosis. Therefore, we started a literature search for thromboprophylaxis-preventive ingredients.

Prof. Yasuhide Yanagisawa

Through literature search, we found and began to focus on nattokinase as an effective thrombolytic agent against thrombosis. At the Chiba Institute of Science, we contacted Prof. Yasuhide Yanagisawa, who researches active ingredients in foods, including nattokinase.

Impetus and Defining Purpose

We focused on nattokinase, which is found in traditional Japanese foods, as a solution for thrombosis. We planned to produce nattokinase using B, subtilis Natto, the bacterium that produces nattokinase. However, there is a lack of prior research on producing nattokinase using B. subtilis Natto, and it is difficult to predict the results and potential drawbacks through literature research alone. In addition, we wanted feedback from a researcher on our study. Thus, we consulted Prof. Yanagisawa, who researches nattokinase.

Two-Way Communication

Prof. Yanagisawa gave us positive feedback on our idea to use B. subtilis Natto to produce nattokinase and to focus on the thrombolytic properties of nattokinase in developing our product. He noted that B. subtilis Natto has been consumed by humans for centuries, ensuring its safety.

Analysis

Initially, we planned to extract nattokinase produced by B. subtilis Natto for use. However, we realized that the advantage of using B. subtilis Natto lies in its safety, which is based on years of research and consumption experience, and extracting nattokinase would not fully utilize this benefit.

Implementations

For further implementations in our project, we decided to revise our plan to move away from extraction and explore the options of directly using B. subtilis Natto. In the next section on surveys, we gathered consumer opinions on this approach.

Conducting a Questionnaire to our Target Market

Human-Practice questionnaire survey.pdf

As a part of deciding our plan for the project, we decided to implement our audience, and future target market to improve our plans and create a better suitable product for the market that desires our product. As our research is based on blood-clot-related illnesses, we formulated questions to understand how the respondents acted daily on preventing thrombosis. In addition to that, we also decided to add in elements on genetically modified food and synthetic biology, to grasp a better understanding of how normal citizens think of the word, and what stigma is existent.

Two-Way Communication

As many common questionnaires represent one-sided communication with the audience, we tried to enhance it, to make it beneficial for both our team and the respondents. For instance, we didn’t just ask questions throughout the form but also focused on giving precise information to answer the question, providing links for unfamiliar and complex terms used in synthetic biology. Having had 54 people answer our questionnaire, we additionally asked them to spread the word about our project on their social media accounts and school/workplaces to promote the further spread of information and interest in synthetic biology.

Furthermore, we added our E-mail Address and social media links in the description of the questionnaire, in case any respondents take up an interest in our project or synthetic biology. By including information and contact addresses, we essentially tried to support the midget of interests that might have sparked in the respondents of the questionnaire, helping the full extent of their questions and inquiries.

Analysis

As the information from the questionnaire (the overall summary of the questionnaire is included in the pdf above), we can conclude a few points, as well as identify how it has influenced our formation in our project. It was identified that only a small amount of people are putting their work into preventing blood-clot-related illnesses, both from dietary and exercise aspects, which is why our project is trying to identify a new and easy way for users to be able to prevent thrombosis, with just a simple action, instead of having to search for a preventative way themselves.

Furthermore, many respondents felt resistant to genetically modified foods. As genetically modified foods are not often seen on the market in food and agriculture, we can conclude that unfamiliarity is a part of the cause of lacking reliance on genetically modified foods. However, astonishingly many respondents felt less resistant to food produced by B. subtilis Natto compared to food produced by E. Coli Bacteria. Comparing the two data, it could be observed that 28 respondents (n=54) said they either “Feel strongly against it” or “Feel somewhat against it” for food that is implemented with E. Coli Bacteria, which is over half of the total amount of respondents. On the other hand, 4 respondents (n=54) said they either “Feel strongly against it” or “Feel somewhat against it” for food that is implemented with B. subtilis Natto, which clearly shows that there is a significant decrease in respondents’ resistance to food implementing B. subtilis Natto compared to E. Coli Bacteria.

Implementation

Taking this information collected directly from potential consumers, we implemented it in our plan for future improvements. It can be concluded that there is a lack of reliance on genetically modified food from the audience and target market no matter what, and it is essential to consider ways where we can make a reliable and trustworthy product for the consumers. The previous analysis that B. subtilis Natto seems to have a ‘safer’ and better image when implemented food compared to food produced with E. Coli Bacteria reestablished our project and how we are attempting to use B. subtilis Natto to create genetically modified food that is easily accessible and widely accepted. What’s more, the analysis revealed that there is only a small portion of the respondents are taking action to prevent blood-clot-related diseases, which further pours into our motivation in our plan to create a product that easily allows the prevention of thrombosis.

Prof. Su’estugu from Rikkyo University, Prof. Asai from Tokyo University of Agriculture, Prof. Kadoya from Sugiyama Jogakuen University

We spoke with Prof. Masayuki Su’estugu of Rikkyo University, Prof. Ryosuke Kadoya of Sugiyama Jogakuen University, and Prof. Kei Asai of Tokyo University of Agriculture.

inputs and defining purpose

Dr. Su’estugu is an expert in the development and implementation of genomic editing tools which can be applied to various cells, and is also our primary instructor. Dr. Kadoya specializes in the relationship between microorganisms and our environment and health. Dr. Asai is an expert in the development and use of genome editing tools and gene modification technologies for B.subtilis and B.subtilis subsp. natto. We conducted this interview in order to further our understanding of the technological possibilities and limitations of genetically modifying B.subtilis subsp. natto, as well as brainstorm ideas for implementation.
As we used B.subtilis subsp. natto, a bacterial strain whose engineering technologies are still in the developing phase and is more complex than other organisms like E.coli, we wanted to understand the technologies to engineer B.subtilis subsp. natto, especially the conjugation procedure.
Two-Way Communication
As our activities in Human Practice put a spotlight on mutual conversation and learning experiences, we engaged in the interview without one-sided questions and receiving answers, but rather asking the three professors for advice on our project, and whether or not our project is possible to complete in the limited research period we had.
They noted that while engineering B.subtilis subsp. natto comes with many challenges compared to other strains of bacteria more commonly used in synthetic biology, there are still ways to work with the bacteria. Prof. Asai explained that B.subtilis subsp. natto can be modified by first working with B.subtilis, a closely related bacteria, and then using conjugation to transfer the genetic material to our target strain. They commented on our idea to increase the production of nattokinase in B.subtilis subsp. natto and said that it is feasible, given our research period and technologies at hand. Nattokinase is a fibrinolytic enzyme that is reported to be effective in preventing cardiovascular disease unique to B.subtilis subsp. natto. Dr. Kadoya explained to us that while B.subtilis subsp. natto cannot live within our gut for extended periods, they contribute to creating eubiosis within the gut microbiome. Based on this, we got the idea to make B.subtilis subsp. natto and/or nattokinase easier to consume by adding them to food that is less odorous and more widely accepted globally.
Additionally, Prof. Su’estugu advised us that it is better if the results of our modification are visibly measurable, such as using the halo assay. Prof. Asai agreed to lend us his plasmid and genetic modification technology for our project.

Analysis

We came to the idea of developing a means for easier consumption of nattokinase in addition to increasing its production in B.subtilis subsp. natto in order to enhance the cardiovascular disease preventing effects of natto. Furthermore, we stepped forward and asked Mr. Yano, the faculty member of the Su’estugu lab in Rikkyo University and a former member of Prof. Asai’s lab, to help us with the genetic engineering of B.subtilis subsp. natto.

Implementation

The discussion with the three professors allowed us to have a clearer understanding of the genetic modification procedure of B.subtilis subsp. natto. Our understanding of the necessary steps for engineering was greatly enhanced, enabling us to have a better sense of the research timeline of our project. The interview also gave us further insight into the health benefits of natto, encouraging us to stick to our bacterial strain despite the challenges ahead.

Tsuruya Chemical Industries, LTD

Our initial plan was to add Bacillus natto to the thickening agent. Therefore, we contacted a company that develops and manufactures various sweeteners and thickens, , Tsuruya Chemical Industries, LTD. Online, we were able to speak with Mr. Minoru Nishida, who has been involved in the development of care foods for the elderly for many years.

Impetus and Defining Purpose

Initially, we focused on a thickening agent for elderly people with swallowing difficulties as a food that they consume daily, and we were thinking of turning this into a product. Therefore, we consulted with Tsuruya Chemical Industries, Inc. to get feedback from experts in the development and manufacture of thickening agents on the actual form of the thickening agent and suggestions for adding Nattokinase to it.

Two-Way Communication

Mr. Nishida made two points regarding the proposal to add Nattokinase to the thickening agent.

The first is that the idea of adding a health ingredient to a thickening agent is not common. This is because many nursing homes have limited budgets. According to Ms. Nishida, elderly care facilities have a fixed daily food budget, and the average nursing home in Japan only has an average of 700-800 yen per day to spend on food, so it is considered difficult to spend costs even more on thickening agents, even if they are a health necessity product.

Second, the target audience is not clear. For example, there are many different types of “elderly at risk of heart disease,” including low-income groups living on pensions, those interested in health foods, or those with other chronic diseases. It was pointed out that the optimal product would depend on the characteristics of the target population.

He also taught us the importance of narrowing down the target elderly population and finding out what kind of food they usually eat. To do this, he advised that we need to visit hospitals and nursing homes to learn more about the conditions and needs of those places.

We were also given suggestions on the idea of adding Nattokinase to tea leaves and furikake.
We received positive feedback in the direction of making it a food that can be consumed daily.

Analysis

This discussion went beyond our original objectives and provided an opportunity to gain significant insights regarding the realization of the product. We realized that our intended target audience did not match the demographic that would use the thickening agent. In addition, the lack of clarity about what the elderly eat, as opposed to our goal of “saving the elderly (65+) at risk of heart disease at a reasonable price that anyone can consume,” was highlighted as an issue.
Additionally, they learned that product pricing and the financial situation of the target population need to be taken into account.
We also learned that. this

Implementation

▶ Change of direction from the thickener idea
We felt that we needed to find a product that matched our target group of “elderly people at risk of heart disease at a reasonable price that anyone can consume,” so we took Nishida’s suggestion and visited nursing homes. We decided to observe what kind of foods our target group takes daily.

Care Home Happiness

We visited the elderly care facility “Care Home Happiness” to explore consumer needs to find an optimal product for elderly people at risk of thrombosis, as an alternative to thickeners.

Impetus and Defining Purpose

Through discussions with Mr. Nishida of Tsuruya Chemical Industries, LTD, we realized the need to find a way to provide preventative foods for elderly people at risk of heart disease at an affordable price. To explore what kind of products would allow the elderly to consume nattokinase regularly in their daily diet, we visited “Care Home Happiness” to gather opinions from elderly residents and caregivers.

Two-Way Communication

We discussed this with Ms. Sumire Okamoto, a manager at the facility and someone involved in elderly care. We also had the opportunity to observe meals and directly talk with the elderly residents.

Ms. Okamoto informed us that natto is not served at the facility since the sticky texture of natto makes it difficult to prepare, especially for elderly residents with swallowing difficulties, as it cannot be offered in a pureed or minced form. Additionally, Ms. Okamoto suggested that our product could be beneficial not only for the elderly but also for middle-aged adults. While conditions like strokes and heart attacks are common among the elderly, middle-aged people also need to be aware of these risks. Ms. Okamoto believed that while the idea of creating a product that “elderly people commonly consume” is good, it should not be limited to something only the elderly consume, like thickeners.

During conversations with the elderly, we learned that although natto is not provided at the facility, many residents expressed a desire to eat it for health reasons. There was also interest in a product that could help improve blood circulation.

Analysis

1. The fact that there are no opportunities to eat natto in nursing homes highlights the importance of our project. It presents a chance to deliver the health benefits of natto to people who currently lack access to it. From our observations, we realized that the eating habits of the elderly are not that different from ours.
2. Ms. Okamoto’s suggestion that our product should be suitable for all age groups, not just the elderly, had a big impact on us. We learned that in the nursing home, tea and oral rehydration solutions are regularly provided for hydration. Tea is consumed by people of all ages worldwide and is commonly offered in elderly care facilities. Based on this, we concluded that tea would be an ideal product for delivering nattokinase in a way that could be consumed daily.
3. We faced challenges when speaking with the elderly. Some had Alzheimer’s disease, which made it difficult to gather detailed opinions. While we could ask about their preference for natto and how often they eat it, discussing topics like synthetic biology, their awareness of thrombosis, or their thoughts on genetically modified foods was challenging.

Implementation

We decided to develop our product as green tea, since it is something that the elderly consume regularly and can be consumed by people of all ages, regardless of where they live.

In our educational activities, we focused on reaching all age groups but placed particular emphasis on engaging middle and high school students. This is because if people do not learn about synthetic biology while they are young, they may develop resistance to new knowledge, making it harder to learn later. Additionally, it was difficult to discuss genetic modification techniques with the elderly, so we felt it was important to start educational efforts with younger generations.

For more details, see Education wiki

Hiroshima City Asaminami Ward Office Food Guidance Division

We consulted the Hiroshima City Health Department’s Food Guidance Division to learn about the country’s laws and policies regarding health foods. We had the opportunity to speak with Ms. Runa Matsuura from the Food Monitoring Section and Ms. Mariko Ishikawa, an administrative instructor from the Broad Area Food Section.

Impetus and Defining Purpose

To implement our project, we reached out to the Hiroshima City Health Department’s Food Guidance Division to understand the national system and verify if there are any legal measures or restrictions in place. In Japan in 2024, there have been cases where multiple individuals suffered health issues due to the consumption of health foods, raising concerns about their safety. Therefore, we believe it is essential for us, as we establish our health food project, to deepen our knowledge about safety and legal regulations.

Two-Way Communication

We visited the Hiroshima City Ward Office and had a discussion with Ms. Runa Matsuura from the Food Monitoring Section and Ms. Mariko Ishikawa from the Broad Area Food Section. As a result, we understood the following points:

There are three classifications of products referred to as “health foods” under national law:

- Specific Health Foods (Tokuho): These are reviewed for safety and efficacy by the government, and the Consumer Affairs Agency grants approval for individual products based on scientific evidence that they can contribute to maintaining or improving health.

- Nutritional Function Foods: If they meet the government-defined standards for ingredients and their amounts, they can display their functions.

- Foods with Functional Claims: This system allows businesses to declare functionality at their own responsibility by notifying the Consumer Affairs Agency about safety and functional information before selling the product.

To ensure the safety of health foods, the Food Guidance Division conducts factory inspections and provides information.

We also discussed regulations and labeling methods for genetically modified foods.

Analysis

This discussion was extremely meaningful for us as it clarified the path towards implementation. Based on the materials and information we received, we learned that our product falls under the classification of “Specific Health Foods.” Additionally, the discussions about genetically modified foods highlighted that our understanding of the Cartagena Protocol related to food is still insufficient.

Implementation

Following this discussion, we are considering further inquiries into the regulations regarding the use of genetically modified ingredients in food. We also gained a deeper understanding of the regulations surrounding Specific Health Foods, and we collectively recognized the need for a government review when we reach the implementation stage.

Prof. Yamamoto From Hiroshima University

We spoke with Prof. Taku Yamamoto of Hiroshima University -an expert in the development and use industry of genome editing tools and gene modification technologies that can be used in a wide variety of cells and organisms, and a member of the national Cartagena Law’s committee on genome editing technologies- to have a better understanding in the rules and borders of the Cartagena Protocol on Biosafety to the Convention on Biological Diversity.
As we are producing genetically modified food products, we wanted to ensure the safety and rules, so we interviewed Prof. Yamamoto to have a clearer view of the biological safety laws. It is an understatement to say that not understanding the safety laws while producing a genome-edited food product by ourselves is extremely dangerous, which could lead to future consumers seeing the product as unsafe and unreliable, to prevent those stigmas at all costs, we have decided to take up an opportunity to learn the safety laws implemented by the government and officials.

Two-way Communication

As our activities in Human Practice put a spotlight on mutual conversation and learning experiences, we engaged in the interview without one-sided questions and receiving answers, but rather asking Prof. Yamamoto for advice on our project, and whether or not our project would pass the examinations and laws of safety if implemented as a food product in the future.
He noted that although there are laws and rules, especially the Cartagena protocol, it is extremely difficult to define the ‘rights’ and ‘wrongs’, and instead there is a big portion of grey space that differs from case to case. Explaining our project, he added that it would be difficult to identify the B. subtilis Natto as self-cloned if there were other genes added by human hands that were non-existent in the original B. subtilis Natto. However, this applies mostly to E. Coli bacteria-produced genes, and it is rarely put in the case of B. subtilis Natto’s promoters and it is difficult to define if it is self-cloned or not.
Especially in Japan, the Cartagena laws are stricter compared to other areas around the world, and some extremists oppose the ideology of genetically modified food. Under this circumstance, it is extremely difficult to persuade consumers about the safety of genetically modified food and ensure 100% of its safety as well. Therefore, paper documents approved by officials and the government proving that the genetic modification was done safely under the Cartagena Protocol, evidence that safety was checked, and evidence that the protein that was the final goal was achieved must be shown to convince and officially prove the safety of the product.

Analysis

Taking in the advice given by Prof. Yamamoto, we stepped forward and asked Mr. Yano, the faculty member of Rikkyo University in charge of our project, to see if our gene would be considered self-cloning. There, It was found that the genes in the phage used to prevent the influx and the drug resistance genes in the plasmids used were not genes originally possessed by B. subtilis Natto, so they will not be categorized as self-cloning.

Implementation

We struggled to experiment using B. subtilis Natto which had not been genetically modified as it had not yet developed as a host and did not contain drug resistance. However, in the end, we still considered experiments without drug-resistance genes or phage in order to make sure that the implementation would be strictly self-cloning, making the product reliable and trustworthy to the consumers while being a genetically modified food.

Planning for implementation

This time, due to the relationship between the plasmid we received and the fact that the technology for genetic modification of Bacillus subtilis natto was not yet developed, we were not able to do it, but we never stopped thinking thoughtfully about how to make it happen. In our project, the prevention of horizontal gene transfer (HGT) and the establishment of biological containment is crucial for safe implementation. We propose several specific methods to mitigate HGT and ensure effective containment.

Prevention of Plasmid Transfer (Conjugation)

Conjugation Mechanism and Control: Conjugation in Bacillus subtilis plasmids, such as pLS20, requires specific genes to express membrane-associated structures known as transferosomes. These structures facilitate the transfer of single-stranded DNA to recipient cells. Recent studies indicate that conjugation is regulated by a plasmid-encoded repressor that keeps the system in an “OFF” state until a specific signal activates it(9). By modifying the genetic circuit responsible for conjugation to make it strictly regulated or inducible only under specific conditions, we can significantly reduce the risk of plasmid transfer to unintended environments.

Risk Assessment of Phage-Mediated Transfer

Phage SPP1 and DNA Transfer: Bacillus subtilis can be infected by phage SPP1, which has been shown to facilitate horizontal transfer of plasmids and chromosomal DNA. This process involves several phage-encoded proteins that are essential for DNA replication and transduction(10). Disruption of these phage proteins or engineering the plasmid to lack recognition sites required for phage-mediated DNA packaging could reduce the likelihood of gene transfer. Additionally, using nuclease-deficient strains can further reduce the chance of plasmid establishment in recipient cells following potential phage infection.

Biological Containment Strategies

Auxotrophy (Nutritional Dependency): Creating an auxotrophic Bacillus subtilis strain that requires an external supply of a specific nutrient only present in the human gut environment ensures that the bacteria cannot survive outside the intended area. Research has shown that this approach is feasible through targeted gene knockout strategies, where metabolic pathways are disrupted, rendering the bacteria dependent on externally supplied nutrients. This method has been successfully applied to other bacteria in laboratory settings and can be adapted for Bacillus subtilis.
Kill Switch Implementation: A kill switch system can be introduced into the bacteria, programmed to trigger self-destruction under certain environmental conditions. For instance, introducing a genetic circuit that induces toxin production upon exposure to light or temperature changes (e.g., above or below 37°C) can ensure that the bacteria only survive within the controlled environment of the human body. Existing kill switch designs, including those responsive to temperature, chemical inducers, or light, have been demonstrated in various microorganisms(9).

Japan Meetup

As a Highschooler iGEM team located in Japan, we have joined the iGEM Japan Community, which consists of not only iGEM teams from the high school, undergrad, and overgrad divisions who are participating this year but also their alumni and past judges who are willing to support us and give us advice on our project.

For the meetup in March held both face-to-face at Tokyo Institute of Technology and online, as it was early on into the iGEM progress, each team took their time to present their pre-made PowerPoint on their plans, problems they are trying to overcome with their project, and future planning schedules within a 5-minute time limit, followed by a Q & A session. As most teams consisted of university students other than our team, we had a fruitful harvest of advice and feedback from other iGEM teams, as well as a chance to ask questions and interact with staff from the headquarters of iGEM who was visiting Japan and stopped by to provide us with a deeper understanding of iGEM and the competition itself. We can conclude that this event provided an effective and motivational start in the iGEM Journey, being able to interact and learn from other teams, in a hybrid format, which widened the possibilities of interactions despite the distance that may have been a barrier in the past when meetups were limited to face-to-face.

Reference

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3. World Stroke Organization. “Global Stroke Fact Sheet.” World Stroke Organization, 2022. Accessed 2 Oct. 2024
4. WHO. “The Top 10 Causes of Death.” Who. int, World Health Organization: WHO, 7 Aug. 2024. Accessed 2 Oct. 2024.
5. Ministry of Health, Labour and Welfare. “令和５年(2023) 人口動態統計月報年計(概数）の概況” (Overview of the Monthly Report on Vital Statistics for 2023 (Provisional Figures)), 2023, Accessed 2 Oct. 2024
6. Ministry of Health, Labour and Welfare. “平成22年国民生活基礎調査の概況” (Overview of the 2010 National Lifestyle Survey), 2010, Accessed 2 Oct. 2024
7. Toshiharu Gochi, et al. “Cost of Illness of Ischemic Heart Disease in Japan: A Time Trend and Future Projections.” Environmental Health and Preventive Medicine, vol. 23, no. 1, 24 May 2018. Accessed 2 Oct. 2024.
8. McMechan, S. R, and A A J. Adgey. “Age-Related Outcome in Acute Myocardial Infarction.” BMJ, vol. 317, no. 7169, 14 Nov. 1998, pp. 1334–1335. Accessed 2 Oct. 2024.
9. SINGH, Praveen K., et al. Mobility of the native Bacillus subtilis conjugative plasmid pLS20 is regulated by intercellular signaling. PLoS Genetics, 2013, 9.10: e1003892.
10. VALERO-RELLO, Ana, et al. Molecular mechanisms that contribute to horizontal transfer of plasmids by the bacteriophage SPP1. Frontiers in Microbiology, 2017, 8: 1816.