HP Value Revisit ​

Before diving into the implementation phase, it's important to revisit the comprehensive Human Practices (HP) values that guide our project. These principles ensure that our approach remains grounded in safety, innovation, and societal impact.

1. Investigating the knowledge gap in coral synthetic and adopting a comprehensive approach to biosafety considerations

2. Developing an synthetic biology approach to address coral bleaching

3. Introducing a novel to expand the coral farming industry and boost economic benefits

4. Ensuring our design considering balance between diverse needs of various coral-related

5. of corals, coral bleaching and conservation through various outreach activities

The future implementation of our project will be firmly rooted in these guiding principles, ensuring that every aspect of our project adheres to ethical standards, scientific rigor, and societal needs, which echoes the "" and "" for human practices.

Pre-Implementation Safety Consideration ​

To responsibly implement our synthetic biology project, it is critical to ensure that we follow the existing biosafety regulations. The consideration in biosafety closely aligns with our : Investigating the knowledge gap in coral synthetic biosafety and adopting a comprehensive approach to biosafety. Since there is no specific guidelines for the release of genetically modified organisms in China, we try to adhere to a strict framework for safety approval according to the EPA guidelines for the release of genetically modified organisms (GMOs), from which we extract three aspects for safety requirements.

1. Ecological Impact Assessment ​

To ensure the safe release of our genetically modified bacteria into coral ecosystems, we must conduct a thorough ecological impact assessment in advance. Specifically, we will focus on several aspects,including disruption of population balance, interference with evolutionary processes and environmental contamination, which will be further discussed in detail in the concern part in our first implementation of coral conservation. Here we only demonstrate our assessment strategy to meet the biosafety requirement.

• Disruption of Population Balance: Controlled co-cultivation experiments with natural coral microbiomes will monitor interactions between GM bacteria and native microbes under stress conditions, such as temperature and light. Molecular techniques like qPCR and 16S rRNA sequencing will track shifts in microbial populations, while metagenomic sequencing will analyze changes in microbial diversity. The study will quantify the competitive advantage of GM bacteria and evaluate their potential to outcompete native microbes, affecting overall coral health.
• Interference with evolutionary processes: This assessment focuses on the potential for horizontal and vertical gene transfer (HGT and VGT) between GM bacteria and local microbial populations. Lab experiments will detect HGT through plasmid exchange or synthetic gene transfer under various conditions, using genetic markers and reporter genes. VGT will be studied by monitoring GM bacteria's ability to transfer genetic material to offspring or host cells. Longitudinal monitoring using high-throughput sequencing will track genetic changes over time in both lab and semi-field environments, providing insights into evolutionary impacts on microbial communities.
• Environmental Contamination: The risk of GM bacteria spreading beyond target reefs will be evaluated through controlled dispersal studies in semi-enclosed reef environments. Tracking the movement of GM bacteria using fluorescent markers, researchers will assess how far and fast they spread. The effectiveness of engineered quorum sensing and suicide switches will be tested to limit bacterial proliferation. Long-term monitoring, using metagenomic sequencing or PCR, will evaluate the persistence of GM bacteria in the environment and their potential to establish in non-target ecosystems, identifying contamination risks.

2. Documentation ​

Following the guidelines from the EPA, it is essential to maintain detailed and accurate regulatory documentation throughout the entire process, especially after conducting the safety assessment testing.

• Project Description: We will prepare a detailed dossier outlining the circuit design, including information on the design and function of the light sensor, thermosensor, and aeBlue chromoprotein. This description will also cover the rationale for using these components in coral conservation and their expected environmental impact.
• Risk Assessment Report: The risk assessment report will include a thorough evaluation of the potential ecological risks. This will encompass the likelihood of gene transfer, effects on other marine speices, persistence in the ocean, and any unintended consequences of the bacteria's release into the ocean. We will also document the proposed containment measures, such as suicide switches or physical protocols, that help minimize the risk of spread outside the coral ecosystem.

3. Social Licensing ​

Gaining public approval, or social licensing, is critical for the success of our project. As we prepare to introduce genetically modified organisms into the environment, we must ensure that the public is well-informed and supportive. To achieve this, we will interact with the public in diverse ways, as shown in our :** Enhance public understanding of corals, coral bleaching and conservation through various outreach activities.

• Public Education Campaigns: We plan to launch outreach efforts to educate the public on the purpose and safety of our project. By clearly explaining how synthetic biology can help protect coral reefs, we aim to address misconceptions about GMOs and reduce resistance to our work.
• Engagement with Stakeholders: Engaging directly with key stakeholders, including local fishing communities and government agencies, will allow us to incorporate their feedback and concerns into our project design. This participatory approach will help foster transparency and build public trust.
• Clear Communication: It is vital to present clear, fact-based information about the benefits and risks of our genetically modified bacteria. By maintaining transparency and being responsive to public concerns towards our engineered bacteria, we aim to secure the necessary social license for environmental release in the ocean.

By addressing these three critical aspects, we can ensure that our project is implemented responsibly in the ocean ecosystem, in accordance with the highest standards of biosafety and public engagement.

Our Expectation in Biosafety Regulation ​

As a China-based iGEM team, we aim to prioritize local regulations when addressing biosafety concerns, ensuring compliance with national guidelines. However, the process has not been as straightforward as we anticipated. During our research, we discovered that China lacks comprehensive guidelines for the environmental release of genetically modified organisms. Existing regulations are primarily focused on agriculture, specifically plants, with no clear and actionable standards for engineered bacteria or the application of synthetic biology in marine environments. This has presented significant challenges in our investigation. We sought assistance from the South China Sea Institute and Guangdong Ocean Laboratory, but experts confirmed that the current regulations are vague and not well-defined. Therefore, and call for more efforts and updates from relevant authorities to establish clearer and more robust guidelines.

Implementation ​

After thoroughly discussing safety concerns, we can now introduce the two key application scenarios for our project: coral conservation and offering a new coral strain to the market. As a responsible team, we have carefully reflected the application background and process in both scenarios, identifying our current limitations and concerns, and responding with reasonable, accountable solutions. This reflects our commitment to implementing the project in line with the 3R principles. Notably, by adhering closely to : Ensuring our design considers the balance between the diverse needs of various coral-related stakeholders**, we strive to make our considerations as comprehensive and practical as possible, predicting risks and offering solutions that maximize benefits for all stakeholders. This paves the way for successful project implementation that is beneficial to all.

Implementation 1: Coral Conservation ​

The first step of our project focuses on coral conservation, which aligns with : Developing an innovative and effective synthetic biology approach to address coral bleaching.

Since our project has only undergone preliminary testing in E. coli, more efforts are required before it can be applied in real-world scenarios. The general pathway for further testing can be summarized as follows:

1. Refining the circuit design and constructing expression vectors: We will finalize the circuit design and test it in E. coli to ensure it functions as expected.
2. Describing circuit behavior in target bacteria: Our current modeling is based on E. coli, but the ultimate application is in Endozoicomonas. This requires us to investigate whether our biobricks function in the target bacteria and perform more precise characterization to build accurate mathematical models.
3. Cocultivation with Coral: We will test the performance of our engineered bacteria in combination with Coral to assess whether it can improve the survival of algae under stress conditions, a key factor in enhancing coral resilience.
4. Lab-based coral testing: In controlled laboratory environments, we will monitor how engineered bacteria affect coral growth and health indicators such as color and growth rate.
5. In-situ testing: Once laboratory testing is successful, in-situ trials will be conducted to evaluate real-world performance and ensure that the modifications benefit corals in natural ecosystems.

After completing the effect testing, we are now ready to apply our engineered bacteria to protect coral systems. To ensure maximum safety and efficiency, we have developed a protocol that prioritizes biosafety and environmental sustainability, shown in the PDF file below:

Limitation ​

• Biomass**:** During our visit to the Hong Kong branch of the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), we recognized a significant challenge: the biomass of our expressed proteins may be insufficient to effectively alter the light environment or mitigate coral bleaching.This issue cannot be definitively verified without conducting examination experiments on corals, which indicates the importance of effect testing experiments. Although time and experimental constraints make such verification experiments currently impossible, we focus on modeling work in our dry lab to ensure that the expression levels align with real-world conditions. More importantly, we are proactively addressing this limitation and have a detailed plan for future testing procedures.

Concerns ​

When deploying our genetically modified bacteria in real-world settings, where they will serve as synthetic probiotics for coral reefs, we may encounter common challenges associated with releasing GMOs into the natural environment:

• Disruption of population balance: Our GMOs, designed to express a light-blocking protein that protects corals from intense light, may have a competitive advantage over other microbes, especially during stressful conditions. This advantage could lead to overpopulation, disrupting the microbial community balance in coral reefs. Given that coral reefs are complex ecosystems, any imbalance in symbiotic microorganisms could further deteriorate coral health.
• Interference with evolutionary processes: Microorganisms frequently engage in horizontal and vertical gene transfer. Our GMO could potentially exchange genetic material with local Endozoicomonas populations, altering the gene pool. Horizontal gene transfer may also lead to the unintended spread of synthetic genes to other species, thereby affecting the local evolutionary process.
• Environmental contamination: Since our bacteria have some mobility and can protect stressed corals, there is a risk that they could spread beyond the targeted reef and establish populations in other coral reefs. Given the parasitic nature of Endozoicomonas, it would be challenging to eradicate them. This raises concerns about long-term environmental contamination from the GMO.

In contrast, if our GMO is too weak, the potential risks mentioned above would be minimal, though it would indicate that the project failed to meet its goals. In such a case, biosafety concerns would be negligible.

These three issues are potential risks in future applications. However, given the unique nature of coral reef ecosystems, it is difficult to estimate the likelihood and magnitude of these harms. Some scientists have observed that when probiotics are introduced to coral systems, they expect to detect these probiotics after a period of time, yet often fail to do so, despite observing improved coral health. This indicates that probiotics may be lost from the system, with loss rates varying across species and environmental conditions, although the underlying mechanism is not well understood. This suggests that our GMO may face limitations and highlights the difficulty in predicting biosafety concerns before conducting field trials.

Based on these concerns, we propose three potential solutions:

• Restrict GMO spread beyond the target reef: This solution aims to limit the spatial distribution of our GMO to the targeted coral reef, preventing its spread to other reefs. One approach could involve quorum sensing. This idea, inspired by discussions with Shimen Middle School students, proposes that since our bacteria have higher abundance within corals but lower abundance in the surrounding environment, we could engineer a quorum-sensing system with a suicide switch. This would trigger the death of the GMO when population levels drop below a threshold, preventing its spread across reefs.
• Designing an active suicide switch: In a worst-case scenario where large-scale GMO leakage and uncontrollable proliferation occur, an active, intervention-based solution is needed to control the population. This could be achieved by designing a chemical-inducible suicide circuit. For example, malic acid-based suicide circuits, which have been well-developed, could serve this purpose. The inducing chemical must be environmentally safe, although rapid increases in its concentration could cause metabolic issues. However, compared to uncontrolled GMO proliferation, this solution may be acceptable in certain cases. More detailed discussions should consider the biochemical properties of the specific inducing chemical.

These solutions represent conceptual directions for addressing the issues. Due to project timelines, we were unable to fully develop and implement these ideas. In the future, we will first test the safety designs of our circuits, and upon successful validation, further develop the entire project.

Implementation 2: New Strain on Coral Market ​

Our project also involves the introduction of a new coral strain, as we state in : Introducing a novel coral strain to expand the coral farming industry.**

Through our HP outreach activities in Guangxi, we interviewed business professionals involved in coral farming and gathered valuable insights. First, we learned that blue corals have a higher market demand and economic value compared to other colors, as they are rarer. Second, most coral farmers and marine aquascaping enthusiasts expressed a high level of acceptance for genetically modified coral varieties and showed great interest in such innovations. Lastly, it was noted that more vibrant corals generally have stricter environmental requirements, such as specific light intensity, temperature, and water quality. This makes large-scale cultivation of vibrant corals more challenging, resulting in a relatively limited customer base. These insights revealed the potential market applications of our project.

The target protein we use, aeBlue, is a blue chromoprotein, meaning corals inoculated with the engineered bacteria would exhibit a noticeable blue tint. Moreover, the functionality of our circuit enhances the coral's adaptability to light and heat in the environment, making large-scale coral farming more feasible and efficient. This led us to consider the possibility of introducing these modified corals as a new strain into the market, generating economic benefits.

Our new strain would not only satisfy the market's desire for novel coral varieties, bringing greater profits to coral farmers, but also help large-scale coral breeders explore new customer bases while providing existing clients with fresh stock. By cultivating corals with greater adaptability, we could potentially reduce the environmental requirements for individual hobbyists and professional farmers, lowering costs associated with equipment and creating greater consumer and producer surplus.

Of course, while we believe this new strain will inject vitality into the coral farming market and bring substantial benefits, as a responsible HP team, we are also mindful of the potential risks and challenges. To ensure that the introduction of this strain poses minimal risks and negative effects, and to promote its widespread and sustainable development, we have carefully considered the possible market issues and risks. From our current standpoint, we have offered responses to address these concerns early on, helping to identify and resolve potential problems as soon as possible.

Concerns: ​

1.Technical risk in Commercial Applications

In commercial applications, the risks are similar to those in Implementation 1, as the core nature of the project remains unchanged. However, the analysis of potential risks in this context should be framed from a commercial perspective:

• Trait instability: The blue coral trait we aim to create depends on the symbiotic relationship between the modified coral and our GMO, which expresses the blue protein aeBlue in response to specific environmental conditions. Maintaining this trait requires stable symbiosis, meaning that if the GMO is outcompeted by other microbes or cannot survive within coral cells, the blue trait will be lost. This problem is also mentioned by business stakeholders we interviewed in Beihai, Guangxi.

• Cross-contamination among products: The blue color trait originates from a motile and reproductive bacterium. It is conceivable that if the bacterium spreads to other corals in the same aquarium, their colors could change. This poses a problem for ornamental coral aquariums, where different colored corals are often displayed together. The spread of modified GMO could cause all corals to turn blue, diminishing the aesthetic value of the aquarium.

Interestingly, the analysis in Implementation 1 focuses on concerns about the potential negative impact of a strong GMO on the environment, while in Implementation 2, we are also concerned about the negative effects of a weak GMO. Additionally, unlike in Implementation 1, we are less worried about environmental impacts in Implementation 2, as ornamental aquariums are controlled, isolated systems, with minimal risk to natural ecosystems if handled properly. The difference in concerns reflects the context-dependency of risk evaluation.

In Implementation 1, coral symbiosis mechanisms with microorganisms remain poorly understood, making it difficult to evaluate the magnitude of trait instability before field testing. However, for product interference, addressing the potential risks in Implementation 1 could reduce the likelihood of cross-contamination between corals in aquariums.

2. Marketing issues

The market is a complex concept, where consumers and suppliers influence each other in multiple ways, collectively shaping market trends. Therefore, the impact of a new strain entering the market will far exceed our initial expectations, including both positive and negative effects. Given the importance of being cautious and preventing negative consequences, we have carefully considered the potential negative impacts of our product and will attempt to provide reasonable responses from our perspective.

• "Passing Off Inferior Products as Superior": As we know, blue corals are rarer and thus command a higher price. Once our genetically modified strain enters the market, two categories will emerge: natural blue corals and genetically modified blue corals. Through market observation and analysis, it is easy to foresee that the concept of "natural" may be overly emphasized by sellers and used as a justification for higher prices, potentially leading to unhealthy inflation of blue coral prices in the market. Additionally, with the introduction of this distinction, there is a risk that some sellers might disguise genetically modified blue corals as natural blue corals to reap large profits from the price difference.
• Excessive Packaging: For coral farmers, "greater survivability and environmental adaptability" is a valuable selling point, especially for large-scale commercial coral farmers, as it can reduce the cost of environmental maintenance. While individual hobbyists may cultivate fewer corals and have limited space to maintain, meaning their environmental setup is often a one-time investment, the increased survivability of these corals remains an attractive selling point. It can help prevent significant coral loss due to mismanagement or equipment failure. In this context, there is a real risk of excessive packaging and overemphasizing the adaptability of the corals, leading to an unhealthy increase in the price of this strain, even though individual hobbyists may not need to invest excessively for these benefits. This price inflation could disrupt the healthy balance of the coral market, ultimately affecting the reputation and sustainable development of market profits.
• Balance of Interests: According to our research, large-scale coral farmers often cater to clients such as aquariums and tourist attractions, which, due to technical and manpower constraints, cannot cultivate corals continuously. As a result, they rely on purchasing and replacing corals regularly, creating a steady demand. Once our highly adaptable corals enter the market, the reduced cultivation requirements will enable these clients to maintain corals at a lower cost and replace them over longer periods, which is a significant benefit. However, this reduced demand—i.e., a leftward shift in the demand curve—might cause a situation where coral farmers, even if they raise the price of this strain to offset the decreased demand, could still see their total revenue decline, thereby harming their profits. Balancing the interests of all parties in this situation will be a challenge.

Although we are not economics experts, we have conducted extensive research and reflection and have proposed some potential solutions to mitigate these foreseeable issues:

• Through public outreach and educational campaigns, we can increase transparency regarding the principles of genetically modified blue corals. By conveying knowledge about corals and genetic modification in a clear and understandable way, we aim to clarify the concepts and characteristics of both "natural" and genetically modified strains, reducing public bias and preventing the price disparity that could arise from such biases.
• By making the effect testing data publicly available, the adaptability of corals inoculated with the modified bacteria will be transparent and accessible. This will allow consumers to directly understand the strain's characteristics, helping to avoid unhealthy price inflation caused by dishonest sellers excessively packaging and promoting the corals.
• We can develop economic models to simulate and predict the relationship between coral farmers and their customers. By analyzing the supply and demand elasticity characteristics on both sides, we can set appropriate production volumes and pricing strategies to maintain balance in the supply and demand curves, ensuring that all parties' interests are protected while maximizing total revenue.

We can implement the first two solutions simultaneously during the promotion of the new strain to prevent the corresponding negative effects. However, the establishment of an effective economic model may require input from more experienced economic experts. We will communicate our considerations to market regulators when launching the strain, seeking their assistance to maximize profits and minimize negative market impacts. We also acknowledge that our ideas may be incomplete and are open to advice and support from market regulators and economic planning departments.

Conclusion ​

By thoroughly evaluating potential risks and proactively addressing them, we ensure that our implementation is both innovative and responsibly sustainable. Each phase of the project, from coral conservation to the introduction of a new coral strain to the market, demonstrates our commitment to a balanced approach—one that integrates environmental protection, economic viability, and societal needs. Through this comprehensive and well-structured strategy, we aim to create a positive, scientifically grounded, and socially embraced impact, embodying the principles of being "Responsible," "Reflective," and "Responsive."