In the iGEM competition, safety is not just a compliance requirement, but a cornerstone of synthetic biology innovation. Our project aims to modify E. coli biofilm to capture sedimenting Ulva prolifera spores to deal with Ulva prolifera infestation, but we are well aware that any bioengineering can bring potential risks. Therefore, from the very beginning of the project, we integrated safety throughout the Design-Build-Test-Learn (DBTL) cycle, ensuring that lab safety, biosafety, and ethical implications were prioritised every step of the way. We protect our team members, the environment and society through comprehensive training and continuous monitoring. It's not just an obligation, it's our core value - to drive responsible scientific innovation.

Lab Safety

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Before entering is lab, all members of the lab group are required to complete the various lab safety courses on the Laboratory Management Platform at Western Liverpool University and pass the associated exams.

Also, before entering the lab, the lab instructor provided safety training to all members of our lab group. This training allowed us to familiarize ourselves with the lab space and the different equipment within it in order to be fully aware of potential hazards and be alerted to how to prevent them from occurring.

All members wore lab coats, goggles, gloves and masks while performing experiments and followed standard protocols for dress code (long hair tied back, long pants, closed shoes, etc.). Members use aseptic techniques in their experiments to follow laboratory behavioural requirements, including washing hands when entering and leaving the lab, disinfecting lab countertops before and after use, cleaning the lab regularly, and maintaining a neat and sterile environment. All chemical reagents used in the laboratory are carefully sorted and stored properly after each use. All members sterilize all wastes generated from microbiology experiments before disposal. Before using any of the instruments, it is ensured that members are fully trained in the operation of the instruments. In the event of an emergency, the Laboratory Manager and the Laboratory Safety Office will be notified immediately.

Project Safety

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Project usage safety: E. coli andUlva prolifera

In our project, we used Escherichia coli as a chassis organism for genetic engineering, and Ulva prolifera and its spores as a model for environmental applications. These materials were selected based on their ease of handling and low-risk properties, but we conducted a thorough risk assessment, referencing iGEM's White List and Check-In Forms, to ensure compliance with 2025 safety standards. Potential risks and our Mitigation Strategy are detailed below, all performed in a BSL-1/2 certified laboratory.

（1）Potential Hazards and Handling of Escherichia coli (E. coli)

E. coli is the main chassis organism for our project and is commonly used to express engineered gene circuits. We mainly use TOP10, BL21, and JM109 type strains, which have a low risk of pathogenicity, but still need to be alert to potential risks.

Potential Risk:

Infection Risk: Although TOP10, BL21, and JM109 strains are generally harmless, accidental ingestion or contact with an open wound may result in minor gastrointestinal distress or infection, especially in immunocompromised individuals. High-risk scenarios include laboratory contamination or accidental release resulting in horizontal gene transfer

Environmental and ecological risk: If the engineered strain escapes the laboratory, it may interfere with the native microbial community or spread antibiotic resistance genes via plasmid transfer.

Laboratory Handling Risks: Aerosols may be generated during incubation, leading to inhalation exposure; chemical reagents (e.g., antibiotics) may trigger sensitization or chemical burns when used adjunctively.

Treatment measures:

Risk assessment and classification: We used the iGEM risk matrix to rate E. coli operations as low to moderate risk (based on BSL-1 criteria). All strains are vetted by Check-In Form with no dual-use concerns.

Laboratory Operations: Strict adherence to Standard Operating Procedures (SOPs), including the wearing of Personal Protective Equipment (PPE) such as gloves, goggles and lab coats. All experiments are conducted in a biological safety cabinet, and culture media are treated using an autoclave. Waste is autoclaved and then sorted and discarded according to laboratory guidelines.

Training and Monitoring: Team members complete online biosafety training and conduct weekly safety debriefings.

Release Prevention: In compliance with iGEM's “Do Not Release” policy, all engineered strains are equipped with a suicide system to ensure that leakage of bacteria is avoided during future use in the field environment, as discussed in the following section.

（2）Potential Risks and Handling Measures of Ulva prolifera and its Spores

Ulva prolifera is a marine green algae that we obtained from Qingdao laboratory.

Potential Risks:

Health risk: Ulva prolifera may carry marine pollutants (e.g., heavy metals or pathogens), which may cause skin irritation or allergic reactions upon contact. Spores can be air or waterborne, leading to inhalation exposure and potentially causing respiratory distress.

Environmental and Ecological Risks: Accidental release of spores into natural water bodies may lead to overpopulation of Ulva prolifera (e.g., green tide outbreaks) and disruption of local ecosystems or agriculture (e.g., blockage of waterways).

Laboratory Operational Risks: Spores are easily dispersed during incubation (e.g., by aerosol or water splashing), increasing the chance of cross-contamination; high humidity environments may promote mold growth.

Treatment Measures:

Risk assessment and categorization: Ulva prolifera handling is rated as low risk (BSL-1 level), but spore handling is elevated to medium risk. We refer to the Laboratory Biosafety Manual, 4th edition, for a SWOT analysis (Strengths: Ease of cultivation; Weaknesses: Spore dispersal).

Laboratory operations: Experiments involving seaweed and Ulva prolifera spores were operated in specific environmental science laboratories at Xi’an Jiaotong-Liverpool University, and all Ulva prolifera and spores were cultured in closed incubators to avoid open exposure. Waste was disposed of by autoclaving or chemical treatment (e.g. ethanol soaking) in compliance with environmental regulations.

Training and monitoring: The team receives specific algae handling training, including a spore safety module.

Project design safety: Light suicide

In our project, safety is not confined to laboratory risks but also extends to social acceptance, ethical implications and policy frameworks. Currently, synthetic biology is confronted with public misunderstanding and regulatory restrictions. We aim to build a safer open application framework by introducing an innovative blue-light suicide control system. The following is our systematic analysis and solution for the application of synthetic biology

1. Public and government attitudes towards synthetic biology: Backward status quo

Although synthetic biology has great potential, the lagging awareness of the public and the government hinders its development. When we were conducting Human Practices activities, we found that most stakeholders were concerned about the unknowns of synthetic biology.

(1) The general public: Afraid because of a lack of understanding.

The general public often lacks understanding of synthetic biology and associates it with "genetically modified monsters" or environmental disasters, leading to fear. For instance, in our initial research, some stakeholders raised the question: What if the modified microorganisms accidentally escape from the hardware, affecting the growth of nori and the environmental ecology, and thereby modifying the human genome?

(2) The government: It is difficult to implement due to its concern for the public. due to its concern for the public

The government gives priority to public opinion. To maintain social stability, it will not overly promote synthetic biology. Take China as an example. The government strictly regulates through the Biosecurity Law, but decisions are often based on conservative risk assessments rather than the latest scientific evidence. This leads to the application of synthetic biology being mostly confined to laboratories or factories, restricting innovation.

2. Synthetic biology requires a larger stage

To overcome these obstacles, we emphasise the transformation of synthetic biology from closed to open.

(1) Current situation: Trapped in the factory

At present, synthetic biology is mainly limited to industrial factory environments, such as cultivating the extracted products of engineered Escherichia coli in fermentation tanks. Although this approach can prevent the release of risks, it also limits the potential that synthetic biology originally had.

(2) Future: Moving towards an open environment

We hope that in the future, synthetic biology can directly act on natural ecosystems and provide sustainable solutions. So we proposed a three-quarter value to initially achieve the application of synthetic biology in a semi-open environment. This definitely requires us to make efforts simultaneously in public awareness, biosafety components and hardware design.

3. Our solution: Blue Light Suicide System——Lucia

To achieve safe expansion, we have developed a new type of blue light-controlled suicide system as a biological component and integrated it into the E. coli chassis. This system ensures that engineered organisms self-destruct after leaving the designated environment, in line with iGEM's "Do Not Release" policy.

(1) Principle

The system, based on the photosensitive protein tag LOV and the tetracycline promoter, activates the suicide gene mazF when exposed to specific wavelengths of blue light (500-600nm). When there is no blue light, the tetracycline repressor protein with the LOV tag binds normally to the tetracycline operon and does not express the downstream pathway. When blue light is present, the tetracycline repressor protein with the LOV tag is degraded and cannot bind to the tetracycline operon, normally expressing the downstream mazF suicide gene. For details, please refer to the design page.

(2) Function verification

We verified through laboratory experiments that the survival rate of engineered Escherichia coli drops to less than 1% under blue light irradiation. For details, please refer to the Results page.

(3) Lucia's scientific research value

To explore the acceptance of the new components by the biological staff and ensure that the components truly play a role in the project, we launched a public interaction activity. We designed a questionnaire survey on iGEM members' views on suicide components at CCiC.

First of all, we investigated their feelings about the use of existing suicide components. As can be seen from the figure, they believe that the existing suicide components still need improvement in terms of response speed and safety.

Then, we introduced Lucia's mechanism in detail to them. 87.5% of them would think that the induction conditions of this component are simple and suitable for application in natural water environments. 62.5% of people think that this component is safer than the existing suicide components.

Then, we introduced Lucia's mechanism in detail to them. 87.5% of them would think that the induction conditions of this component are simple and suitable for application in natural water environments. 62.5% of people think that this component is safer than the existing suicide components.

In conclusion, the field of synthetic biology requires more functional and powerful suicide elements. The Lucia we designed has a high acceptance rate among iGEMers. Next, we will further enhance its application value through precise quantitative analysis to provide data support for the practical application of synthetic biology safety components.

(4) Hardware —— CBA

We have designed a small, field-deployable filter hardware that integrates a coarse filtration system and a biological filtration system. We call it CBA —— Compact Biological Abyss. This device not only effectively filters spores from seawater but also ensures the stable attachment and efficient functional performance of engineered bacteria, thereby achieving precise intervention in marine environments. The hardware is divided into two main modules: the coarse filtration system and the biological filtration system. The coarse filtration system includes a filter box and a sand separation device, primarily used for initial removal of adult algae, sediment, and large particulate impurities; the biological filtration system centers on a minimal surface moving bed biofilm reactor (MBBR), which carries the engineered bacteria for spore adsorption. The two modules are conveniently c onnected via magnetic attachment devices, facilitating assembly, disassembly, and maintenance. To enhance biosafety, the entire device adopts a fully dark environment design, ensuring the normal operation of the engineered bacteria's blue light suicide system—once an accidental leak occurs, the suicide mechanism can be immediately activated to prevent potential environmental pollution and ecological risks. At the same time, the overall device is made of PLA material, which is derived from renewable resources such as corn starch and is considered an environmentally friendly alternative to traditional plastics, suitable for marine deployment. We hope that through this safe and reliable hardware platform, the engineered bacteria can better perform their role, promoting sustainable marine ecological governance. For details, please refer to the hardware page.

Project Ethical Safety

To determine whether the project complies with safety and ethical requirements, we specifically interviewed Teacher Shiwen Ma from Suzhou University, consulting her professional opinions on the feasibility and improvement of the microbial suicide system in this project. Ms. Ma affirmed our idea of designing the microbial suicide system, and at the same time pointed out that to truly promote the implementation of this system, it is necessary to establish a supporting and systematic "Comprehensive Management Program for Biosafety in the Natural Environment". Based on this suggestion, we will take this project as a specific case and combine the framework of the comprehensive management program proposed by Ms. Ma to conduct an in-depth analysis of the key aspects that need further refinement in the current microbial suicide system from the perspective of safety management.

I. Module for Ecological Risk Prediction and Biosafety Controllability Construction

1. Core Ecological Risks

1. Risk of Horizontal Gene Transfer: Theoretically, the modified gene fragments may be horizontally transferred to native microorganisms in the environment through methods such as plasmid exchange.

   XJTLU-CHINA: Lucia (the modified bacterial system) is not designed with relevant functions to prevent this, so gene contamination cannot be avoided temporarily.

2. Risk of Food Chain Disturbance and Accidental Ingestion: It is necessary to consider whether marine organisms will accidentally ingest the modified bacteria and the potential impacts of their metabolites.

   XJTLU-CHINA: Hardware and the Lucia system are designed to ensure that the engineered bacteria cannot survive outside the hardware. However, the potential impacts of metabolites cannot be addressed.

3. Risk of Evolutionary Mutation and Resistance: Under environmental stress, the modified bacteria may mutate, which could lead to the loss of photocatalytic self-destruction function or the development of light resistance in theory.

   XJTLU-CHINA: The probability of mutation is relatively low, and we will regularly replace the bacteria with fresh bacterial solutions.

2. Biosafety Controllability

Ensuring the physical and functional controllability of the engineered bacteria is the bottom line of the project design.

1. Physical Controllability: The modified bacteria are restricted by the physical environment and can only function at specific locations.

   XJTLU-CHINA: The bacteria are encapsulated in a light-proof hardware carrier. They can only grow and reproduce normally inside this carrier, and will be quickly inactivated by light once they escape.

2. Functional Controllability: It means that the target functions of the modified bacteria can be precisely regulated—their functional performance can be activated, adjusted, or terminated according to actual needs. At the same time, it can effectively prevent the bacteria from producing unintended functions that cause harm to the environment or organisms, ensuring that the modified bacteria only operate within the designated scope in accordance with the designed functions.

   XJTLU-CHINA: The modified bacteria are extremely sensitive to light and will die directly when exposed to light with a wavelength of 465 nm. They can still express their functions normally under light-proof conditions.

II. Module for Collaborative Prevention and Control of Cross-Border Biological Pollution

The ecological environment is extremely complex; for example, the connectivity of the ocean means that any released substances may spread with ocean currents. Therefore, the module for collaborative prevention and control of cross-border biological pollution is particularly important. We classify cross-border biological pollution into two categories:

1. Passive Cross-Border Spread: Low-probability and unavoidable incidents such as the leakage of modified bacteria. This scenario places higher demands on the self-destruction function of the modified bacteria and the emergency measures of the hardware.

   XJTLU-CHINA: The natural survival time of E. coli (several hours to several days) is much shorter than the time required for ocean currents to carry it to the high seas or the waters of other countries (several weeks to several months). Moreover, the natural light on the ocean surface can serve as an effective secondary inactivation guarantee. For extreme situations such as uneven light exposure and equipment damage, the hardware design has considered redundant lighting arrangements and structural strength.

2. Active Cross-Border Transport: It refers to controllable scenarios where the modified bacteria, their related carriers, and equipment need to be proactively transported to the territory of other countries due to project needs (such as cross-border cooperative research and joint cross-border pollution control).

   XJTLU-CHINA: A joint prevention and control mechanism for cross-border risks is proposed. We can collaborate with customs, environmental protection, agriculture, and other departments to establish a list of cross-border biological pollutants and formulate international standards. In addition, we will complete the safety assessment report of the modified bacteria and obtain cross-border transportation permits in accordance with the biosafety regulations of the target country and international standards.

III. Module for Risk-Benefit Balance and Alternative Scheme Management

Only when it is clearly proven that "benefits outweigh risks" can the application of a technology be ethically justified.

1. Comprehensive Benefit Analysis

This section identifies the practical benefits brought by this technology.

XJTLU-CHINA:

1. Ecological Benefits: Fundamentally reduce or even replace the use of chemical algaecides, significantly reducing marine pollution; effectively control the overgrowth of Enteromorpha (green tide algae) and restore the balance of damaged marine ecosystems.
2. Socio-Economic Benefits: Improve coastal landscapes and enhance the attractiveness of the tourism industry; reduce the economic losses of fishermen caused by Enteromorpha and promote the sustainable development of the fishery industry; in the long run, compared with the 200-300 million yuan annually invested in Qingdao for mechanical salvage and chemical treatment (of Enteromorpha), the long-term operation and maintenance costs and ecological restoration costs of this program will be significantly lower, demonstrating better economic efficiency and sustainability.

2. Comprehensive Risk Analysis

This section identifies the potential risks of this technology.

XJTLU-CHINA:

1. Ecological Risks: There is a possibility of leakage of the modified bacteria.
2. Socio-Economic Risks: This program requires high initial R&D and deployment investment; the large-scale deployment of hardware may hinder the navigation of ships.

3. Comparison with Traditional Methods

This section compares the new program with existing methods to highlight its advantages.

XJTLU-CHINA:

1. Mechanical Salvage: Consumes enormous human and material resources, only addresses the symptoms rather than the root cause, and the subsequent disposal of the salvaged materials is prone to causing secondary pollution.
2. Chemical Agents: Cause severe pollution, harm non-target organisms, damage the ecological balance, and are unsustainable.
3. Biodegradation by Native Microorganisms: Has low efficiency and is unable to cope with large-scale Enteromorpha outbreaks.

4. Conclusion

This section summarizes whether the benefits of the new program outweigh the risks after assessment.

XJTLU-CHINA: The modified bacteria program demonstrates comprehensive advantages in terms of treatment efficiency, long-term cost control, and ecological friendliness. It is a powerful supplement and innovative approach to address the current challenges in Enteromorpha control.

IV. Module for Regulatory Compliance and Public Participation Guarantee

1. Regulatory Compliance

   Conduct self-inspections and accept third-party inspections to verify the project’s compliance with laws, and issue compliance statements.

   XJTLU-CHINA: All laboratory research of this project is conducted in laboratories that meet the corresponding Biosafety Level (BSL) requirements, in accordance with China’s Biosafety Law, Marine Environmental Protection Law, and Regulations on the Biosafety Management of Pathogenic Microorganism Laboratories. The project has also undergone review by the ethics committees of the university and cooperative institutions.

2. Public Participation Guarantee

   Ensure the public’s right to know and supervise the project, and safeguard the actual interests of the public.

   XJTLU-CHINA: Although the current stage of the competition is focused on program design, we have already incorporated public participation into our long-term planning as an indispensable link. Before any practical application in the natural environment in the future, we must go through the complete government approval process, and proactively hold public hearings, expert demonstration meetings, and public consultation meetings. We will fully absorb opinions from all parties and advance the project cautiously on the basis of reaching social consensus.

V. Module for Emergency Response to Biosafety Out-of-Control Incidents

In response to the risk of large-scale spread caused by the leakage of modified bacteria, it is necessary to formulate differentiated and rapid emergency response plans based on the characteristics of the environment where the leakage occurs. The core idea is "monitoring and early warning + rapid inactivation + physical isolation + cross-departmental collaboration". The specific plan is illustrated with the example of XJTLU-CHINA:

1. Monitoring and Early Warning: The real-time monitoring system is crucial for detecting abnormalities at the earliest opportunity. We install monitoring devices on the hardware and conduct regular sampling and monitoring of seawater.
2. Rapid Inactivation: Once an early warning is triggered, the backup plan will be activated immediately to block off part of the sea area, ensuring the rapid elimination of the modified bacteria within the physically isolated area.
3. Physical Isolation: Isolation membranes are pre-installed in the pilot sea area to prevent the situation from escalating.
4. Cross-Departmental Collaboration: Establish a joint emergency command mechanism with government departments such as marine affairs, environmental protection, and fishery administration to ensure rapid response and effective implementation of measures.

Future Vision

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Looking ahead, we’ll advance the "three-quarter value" concept, develop more open-environment suicide systems, and Lucia variants to boost public acceptance. Via risk assessment, protocols and ethics reviews, we safely explore synthetic biology’s potential, follow safety standards and add improvement mechanisms. We’ll extend these practices to the wider community, raise industry standards and enable safe synthetic biology advancement.