Overview
In line with iGEM's commitment to responsible innovation, safety is a core principle in our project. Given the critical importance of biosafety in preventing unintended harm, our team has applied iGEM's safety principles in project design, lab work, human practice, and risk identification to ensure a comprehensive safety framework.
| Question | Description |
|---|---|
| Did the team make a contribution to biosafety and/or biosecurity? | Yes. The team contributed to biosafety and biosecurity by designing a temperature and blue light-induced suicide switch. We also shared lab safety protocols and emphasized biosafety in Human Practices through education and policy recommendations. |
| Is their contribution well characterized and/or well-validated? | Yes. We have experimentally characterized the designed suicide circuit and optimized the RNA thermometer through modeling. Further characterization work will be conducted in the future, but these components have already been well-characterized in the original publications. |
| Did the team build upon existing knowledge, understanding, tools or approaches? | Yes. Our team built upon existing knowledge and tools. We used established principles to design our suicide switch and optimized RNA thermometer structures. We also expanded on existing lab safety protocols and referenced previous research in our Human Practices. |
| In addition to applied safety work, has the team managed any risks from their project appropriately? | Yes. We identified and discussed biological, chemical, physical, and safety risks, and established corresponding emergency measures. We also referenced existing laboratory risk identification and emergency response protocols to manage experimental safety effectively. |
| Has the team addressed the use of synthetic biology beyond the iGEM competition? | Yes. We have explored potential industrial applications of our project and engaged with stakeholders to discuss broader environmental and societal impacts. We have also considered relevant regulatory frameworks for real - world implementation. |
SAFE PROJECT DESIGN
Choice of a Non-pathogenic Chassis
In the pursuit of a safe and effective biological system for anti - influenza applications, the selection of a non - pathogenic chassis organism is of utmost importance. Our project has chosen Lactobacillus rhamnosus GG (LGG) as the chassis, a decision firmly rooted in its well - established safety profile.
LGG is a strain of lactic acid - producing bacteria that has been extensively studied and utilized in the field of probiotics. Multiple clinical trials have demonstrated its safety and beneficial effects. For instance, a randomized, placebo-controlled trial by Luoto et al. (2014) investigated the impact of prebiotic and probiotic supplementation in preterm infants[1]. The study found that probiotic supplementation, which included strains like LGG, was associated with a significant reduction in rhinovirus infections. Meanwhile, LGG can achieve transient colonization in the respiratory tract of mice (with a colonization amount approximately 10 times that of the GR-1 strain), and can significantly bind to mouse nasal mucosal cells and airway macrophages. It can also significantly reduce the number of eosinophils in bronchoalveolar lavage fluid, the levels of interleukin-13 and interleukin-5 in lung tissue, and airway hyperresponsiveness through preventive intervention. In respiratory-related interventions, it demonstrates superior colonization ability and beneficial effects, meeting the application requirements of the project for anti-influenza in the respiratory tract[2]. This not only highlights the potential of LGG in promoting health but also its safety when administered to vulnerable populations.
The non-pathogenic nature of LGG is further supported by its long - standing use in food and dietary supplements. It is generally recognized as safe (GRAS) by regulatory authorities, indicating that it has a low risk of causing disease in humans. Moreover, LGG does not possess virulence factors commonly found in pathogenic bacteria. It lacks genes encoding for toxins, invasins, or other factors that could potentially cause damage to human cells, animals, or plants. This inherent safety makes it an ideal chassis for our engineered biological system, as it minimizes the risk of unintended pathogenicity or harmful side-effects during the application of our anti-influenza solution.
Selection of Low-Harm Parts: Safety of scFv
A key aspect of our project’s safety design is the deliberate selection of components that minimize potential harm to humans, animals, and plants. The central functional part in this regard is the medi8852/1G01 single-chain antibody fragment (scFv).
As an engineered antibody fragment, scFv is designed with high specificity: it targets and binds exclusively to specific receptors on influenza viruses, with no inherent toxicity or off-target effects on human cells, animal tissues, or plant organisms. Its mechanism of action—neutralizing viruses by "locking" their invasion machinery—focuses solely on inhibiting viral infectivity without disrupting normal biological processes in hosts or the environment.
This specificity ensures that scFv operates as a precision tool against influenza, avoiding the broad-spectrum harm that might be associated with non-specific antimicrobial agents. By prioritizing such a targeted, low-risk component, we further reinforce the safety of the overall system, aligning with iGEM’s principles of responsible innovation and harm minimization.
Substitution of Safer Experimental Materials: Use of Inactivated Viruses
In line with our commitment to minimizing risks in experimental procedures, we prioritize the substitution of hazardous materials with safer alternatives, a key example being the use of inactivated influenza viruses in our studies.
Live influenza viruses pose inherent risks of accidental infection or environmental spread, particularly in laboratory settings where handling and manipulation are routine. In contrast, inactivated viruses—treated through methods like chemical inactivation or heat treatment—retain their structural integrity (including the specific receptors targeted by the medi8852/1G01 scFv) while losing their infectivity. This allows us to accurately study the interaction between the scFv and viral particles, validate the neutralization efficiency of our system, and optimize functional parameters without exposing researchers or the environment to the risk of active viral transmission. This substitution not only safeguards the well-being of team members but also prevents accidental release of viable viruses into the environment, reinforcing our adherence to iGEM’s principles of responsible experimentation and risk mitigation.
Implementation of "Kill-Switch" Mechanisms
To prevent unintended persistence or spread of the engineered biological system—whether in the human body or the environment—, we have integrated dual "kill-switch" mechanisms as core safety controls, ensuring precise and responsive risk mitigation.
- Temperature-sensitive suicide system: Leveraging the pDawn system, along with the MazF toxin and its antitoxin MazE, this mechanism provides dual triggers for safety. If engineered organisms are released into the environment, sunlight (which substitutes for blue light) activates the suicide program, halting their persistence. For potential long-term retention in the nasal cavity, deliberate blue light irradiation initiates self-destruction, ensuring controlled elimination.
- Blue-light-activated suicide system: Leveraging the pDawn system, along with the MazF toxin and its antitoxin MazE, this mechanism provides dual triggers for safety. If the engineered organisms are released into the environment, sunlight (which substitutes for blue light) activates the suicide program, halting their persistence. For potential long-term retention in the nasal cavity, deliberate blue light irradiation initiates self-destruction, ensuring controlled elimination.
These "kill-switch" mechanisms, combined with rigorous safety protocols, embody our commitment to iGEM’s principles of responsible innovation, ensuring the engineered system can be safely contained and neutralized when necessary.
SAFE LAB WORK
General Laboratory Safety
The experiments were conducted in the laboratory located in the School of Public Health of Southern Medical University. Our laboratory is classified as BSL-Ⅱ, in compliance with the General Biosafety Standard for laboratories for causative bacteria of the People's Republic of China[3] and Safety Regulations for Higher Education Laboratory [4].
Based on this foundation, our laboratory has already developed a Laboratory Safety Management Manual(Document No.: SWAQGLSC-2025) in accordance with the relevant legal and regulatory elements of the Laboratories - General requirements for biosafety [5], the Regulation on the Bio-safety Management of Pathogenic Microbe Labs [6], the General Guidelines for Biosafety in Microbiology and Biomedical Laboratories [7], the Regulations on the Administration of Medical Waste [8], etc. The manual clearly defines our laboratory's comprehensive management in terms of organizational management, operation of the safety management system, environmental facilities, key equipment, management of microbial strains (toxins) and samples, disinfection and sterilization, waste disposal, laboratory personnel management, emergency management, and security, etc., hereinafter referred to as the "manual".
Equipment Safety Management
The manual states that, if conditions permit, the following safety equipment is recommended for use: biological safety cabinets, pipetting aids, autoclaves, micro inoculation loop heaters, disposable inoculation loops, screw-capped tubes and bottles, leak-proof containers, disposable Pasteur pipettes, disposable sharps containers, etc. Safety equipment such as autoclaves and biological safety cabinets must be accepted in accordance with requirements before being put into use. During routine use, they should be regularly inspected by authorized technical personnel in accordance with international or national standards, referring to the manufacturer's instructions.
| Biosafety Cabinet (BSC) | The biosafety cabinet (BSC) is specifically engineered for procedures involving infectious materials—such as primary cultures, microbial or viral strains, and diagnostic specimens—protecting the operator, the laboratory environment, and the materials themselves from exposure to potentially infectious aerosols and splashes. Proper use of the BSC significantly reduces laboratory-acquired infections caused by aerosol exposure and prevents cross-contamination of cultures. |
| Pipette assist device | Controls leakage at the pipette tip, prevents contamination at the suction end, and protects the pipette, operator, and vacuum tubing. Easy to use and can be sterilized. |
| Screw cap test tubes and bottles | Effectively prevent aerosol generation, spillage, and leakage; resistant to high-pressure sterilization; reusable. |
| Autoclave | Approved designs effectively heat-sterilise infectious materials to ensure the safety of discarded or reused items. |
| Single-use sharps container | Sturdy, puncture-resistant to prevent accidental injury, and resistant to high-pressure sterilisation. |
| Disposable Pasteur plastic pipettes | To avoid using glassware, they should be placed in disinfectant solution after use and disposed of as infectious waste. |
Waste Management Regulations and Disposal Requirements
To standardize the management of laboratory waste, maintain the normal order of inspection work, prevent the occurrence of accidents, and avoid or minimize the harm caused by infectious or potentially infectious biological agents to laboratory personnel, the environment, and the public, the manual has detailed regulations and disposal requirements for laboratory waste management. The handling of laboratory waste should follow the requirements of the relevant regulations such as the Regulations on the Administration of Medical Wastes [8], Implementation Measures for the Management of Medical Waste of Medical and Health Institutions (Ministry of Health) [9], Medical Waste Classification Catalogue (2021 Edition) (National Health Commission of the People's Republic of China) [10], Medical Waste Packaging, Containers and Warning Sign Standards (Ministry of Environmental Protection and Ministry of Health) [11], and should be properly handled in accordance with the principles of "harmlessness, minimization, and resource utilization."
Emergency Response
The laboratory adheres to the principles of “rapid response, scientific handling, truthful reporting, and accountability” to ensure that incidents/accidents are dealt with in a timely and effective manner. To this end, the laboratory has established a reporting system for unexpected incidents/accidents, which includes incident/accident classification, reporting, handling, accountability, and record-keeping and file management.
Personal Safety Protection
The manual stipulates that laboratory technicians must participate in training related to biosafety knowledge and policies, management standards, standard operating procedures relevant to their work, instrument operation procedures, basic biosafety information, biosafety risks, personal protection, and emergency response principles and procedures for laboratory accidents, routine laboratory disinfection procedures and methods, knowledge and techniques for handling biosafety accidents, and emergency response principles and reporting procedures for biosafety accidents. They must also take exams on biosafety knowledge and skills, and only after passing these exams and obtaining the relevant certificates can they start work. Training and assessment records are kept as part of the training archives.
In accordance with this regulation, all members in wet lab have undergone the basic comprehensive safety training for new laboratory personnel at Southern Medical University and have obtained the relevant certificates after passing the corresponding exams.
Safety Promotion in Human Practice
Public Safety Cognition and Communication
We popularise synthetic biology safety knowledge and collect community concerns about project safety through various forms of public engagement activities:
-
Community Lectures and Science Sessions: Safety-themed lectures are organised for the public of different ages and backgrounds, explaining in layman's language the biosafety concepts involved in the project and answering common questions such as "is genetic engineering technology safe".
- Stakeholder Interviews: Conduct in-depth dialogues with medical experts, environmental scientists, ethicists and policy makers to understand the expectations and concerns of different fields on the safe application of the project.Establish a regular communication mechanism to share the progress of project safety assessment and improvement measures on a regular basis; collate and form a list of stakeholders' concerns, which can be used as an important reference basis for project safety optimisation to ensure that the development of the technology is coordinated with the expectations of the society.
- Questionnaire Survey and Feedback: Collect the public’s perception of the project’s potential risks through online and offline research, and incorporate feedback into safety-strategy optimisation. A tiered questionnaire targets the general public, practitioners, researchers, etc., covering risk perception, acceptance of safety standards, and regulatory suggestions. Based on survey results, improvement plans are formulated and the outcomes are made public, creating a closed loop of “collection–analysis–improvement–feedback”.
- Student-Scientist-Public Collaboration: Organise cross-group workshops to jointly discuss the boundaries and norms of safety application of the project, and form a safety consensus.
- Educational outreach: Develop synthetic biology educational materials for primary and secondary schools to integrate the concept of "responsible innovation" into youth science education.The materials include illustrated books, teaching videos and interactive courseware to cultivate students' safety awareness and responsibility.
- Policy Recommendations: Drawing on the insights gained from our project, we will identify the safety challenges and management deficiencies in the R&D and application of synthetic biology. We will then submit well-considered recommendations on the safety management of synthetic biology to relevant organizations to facilitate the development of standards that better meet practical needs. Our proposal will cover key areas such as the risk assessment process and emergency response mechanisms, supported by concrete cases and data to enhance the policy's operability and forward-looking nature.
- Legislative Reference and Improvement:Taking into account the legislative initiatives of other countries and aligning with the current situation in our own country, we have developed a rational improvement program.
- Scientific safety training: Provide safety training to students entering synthetic biology laboratories, explaining in detail the classification of laboratory biosafety levels, the correct donning and doffing procedures of personal protective equipment, demonstrating the classification and treatment of experimental waste and disinfection norms, helping them build up a scientific and rigorous awareness of safe operation.
Stakeholder interview on safety-related content
| Time | Interviewee | Content |
|---|---|---|
| 2025.5.9 | Did the team make a contribution to biosafety and/or biosecurity? | Yes. The team contributed to biosafety and biosecurity by designing a temperature and blue light-induced suicide switch. We also shared lab safety protocols and emphasized biosafety in Human Practices through education and policy recommendations. |
| 2025.5.11 | Is their contribution well characterized and/or well-validated? | Yes. We have experimentally characterized the designed suicide circuit and optimized the RNA thermometer through modeling. Further characterization work will be conducted in the future, but these components have already been well-characterized in the original publications. |
| 2025.5.17 | Did the team build upon existing knowledge, understanding, tools or approaches? | Yes. Our team built upon existing knowledge and tools. We used established principles to design our suicide switch and optimized RNA thermometer structures. We also expanded on existing lab safety protocols and referenced previous research in our Human Practices. |
| 2025.8.17 | In addition to applied safety work, has the team managed any risks from their project appropriately? | Yes. We identified and discussed biological, chemical, physical, and safety risks, and established corresponding emergency measures. We also referenced existing laboratory risk identification and emergency response protocols to manage experimental safety effectively. |
| 2025.10.1 | Has the team addressed the use of synthetic biology beyond the iGEM competition? | Yes. We have explored potential industrial applications of our project and engaged with stakeholders to discuss broader environmental and societal impacts. We have also considered relevant regulatory frameworks for real - world implementation. |
Our original-designed science popularization picture book
Our team has developed a comprehensive set of 'touchable, visible and playable' responsible safety education materials for children and families. We pack safety experiments into boxes containing disposable gloves, masks, and tiered risk instructions, enabling children to learn about protection before conducting scientific experiments. We have also created AR picture books that reveal the transmission chain of the influenza virus with a simple scan, and which include a family sampling challenge at the end of the story. We have printed respiratory infection prevention guidelines in the form of booklets, which are distributed through short online videos and offline community outreach. These booklets deliver a 'one-page risk map' via dual channels. These boxes, books and guidelines are reaching multiple communities and nearly a hundred families, forming a safety education loop involving children's participation, parental involvement and community linkage. This makes safety education substantial, interesting and effective.
These activities have helped us establish open communication channels to ensure that public safety concerns are fully taken into account.
Social Safety Assessment And Policies
To ensure the broad social acceptance of safety standards, we are actively engaged in promoting the development of a safety framework that involves multiple stakeholders:
Personnel Safety
Before entering the laboratory to participate in activities such as bacterial colouring and laboratory tours, we take great care to educate visitors on laboratory safety rules. We emphasise the importance of wearing protective gear such as masks and gloves as a strict requirement. In order to make more people aware of the importance of flu protection and the significance of our project, we conducted HP activities for various groups, during which we marched in groups and were guided by professional staff to ensure safety.
Summary - Social Responsibility
At the initial stage of our project, we found that the public had many concerns about “whether artificially modified probiotics affect the micro-ecology of the human body”, “the duration of action and potential side effects of antibody spray”, and a small number of people were resistant to it. Although the project has undergone rigorous safety testing, doubts about the safety of its application are still spreading in society, casting a shadow over the popularity of this technology. Therefore, it is important for us to educate the public about the relevant science.
We conducted a questionnaire survey to assess the level of public awareness of the spray, and then we invited immunology experts to conduct a scientific seminar to explain, in layman’s terms, the principles of probiotic modification, the mechanism of action of the antibodies, and the multiple validations of the safety of the project during the R&D process, and to answer questions on the spot. We firmly believe that dismantling technical details in a concise and easy-to-understand way is an effective way to eliminate public concerns and increase acceptance.
At the same time, we also realise that if the product is introduced to the market without adequate publicity, it may trigger a collective panic about “artificial biological products”. Therefore, before the product is officially launched on the market, we will carry out a large-scale education campaign covering communities, schools, and medical institutions. Each copy of the product will be accompanied by a detailed description, clearly labelling the safety range supported by the test data and the actual risk difference compared with traditional influenza prevention and control methods, so that the public can make a well-informed choice.
By integrating safety considerations into the entire process of human practice, we not only ensure the responsible development of the project itself, but also promote the dissemination of a synthetic-biology safety culture in society, laying the foundation for a healthy interaction between technology and society.
RISK IDENTIFICATION & RESPONSE
Biological Risks
| Potential Risks | • Pathogenicity: We are working with bacteria classified at Biosafety Level 2 (BSL-2), which have added virulence factors and meet the criteria for Dual-Use Research of Concern (DURC) Category 2 [11]. Accidental ingestion, mucosal exposure, or inhalation of aerosols may lead to gastrointestinal or respiratory infections.
• Environmental Impact: If the culture medium or solid waste is not properly treated, antibiotic resistance genes may be released into the local sewage microbial community. |
|---|---|
| Measures Implemented |
• All operations are conducted within a Biosafety Cabinet Class II (BSC-II). • Laboratory coats and gloves are worn throughout the experiment. • Cultures are inactivated, and liquid waste is subjected to autoclaving at 121 °C for 1 h 30 min. |
Chemical Risks
| Potential Risks | Hazardous Chemicals:
• SDS-PAGE utilizes acrylamide (a neurotoxin in its monomer form), β-mercaptoethanol (a respiratory irritant), and Coomassie Brilliant Blue stain (contains flammable methanol); • The viral plaque reduction assay employs formaldehyde (flammable, and an irritant to eyes, skin, and the respiratory tract). |
|---|---|
| Measures Implemented |
• Acrylamide solutions are pre-mixed by a qualified supplier to minimize monomer exposure.
• All formaldehyde manipulations are carried out inside a chemical fume hood, and the procurement, storage, use, and disposal of formaldehyde strictly follow the hazardous-chemical management protocols specified in the Manual. |
Physical Risks
| Potential Risks | Equipment Safety
•If a centrifuge rotor fails at high speed, infectious aerosols could be generated. •Door-seal leakage from an autoclave can cause scalding or steam injuries. Electrical Safety •Simultaneous operation of multiple heating blocks and electrophoresis units poses a risk of circuit overload when serial power strips are used. |
|---|---|
| Measures Implemented |
•Each instrument is assigned to a designated custodian who organizes regular maintenance and servicing by its authorized users; any malfunction must be reported for repair without delay.
•Autoclave use is logged, and only trained personnel are permitted to operate the unit. •Scheduled inspections of electrical safety and all laboratory equipment are conducted to identify and eliminate hazards promptly. |
Security Risks
Dual-use research of concern (DURC) describes research that is intended to provide a clear benefit, but which could easily be misapplied to do harm. The possibility that research might be misused, either intentionally or accidentally, is a long-standing concern of science. It can have implications in ethics and wider societal issues, and involves not only research communities and public health, but also donors, scientific publishing and public communication [13]. Therefore, through a meticulous risk assessment of DURC, we are able to design and conduct our experiments to the best of our ability, scientifically understand the inherent risks while avoiding the most significant ones.
Horizontal Transfer of Antibiotic Resistance
Our expression plasmid relies on the ermB gene, which encodes a 23S rRNA methyltransferase and confers resistance to erythromycin. ermB is located on a Tn917-type transposon that retains its transposase and resolvase reading frames; consequently, the entire resistance unit can excise and re-integrate into new DNA.
The human anterior nares provide an environment that could favour such events. Resident Streptococcus mitis, S. oralis and Staphylococcus epidermidis enter a competent state in response to lysozyme, cold shock or sub-inhibitory macrolides—conditions that occur during mild colds or after outpatient azithromycin therapy. If our engineered Lactobacillus rhamnosus lyses or leaks DNA, the ermB fragment might be taken up and integrated by homologous recombination, expanding the pool of macrolide-resistant commensals.
Although we have not yet quantified this risk, in future we will perform a 24 h co-culture mating experiment. Immediately afterward we will replace ermB with a non-antibiotic, auxotrophy-based selection and, in the final version, integrate the scFv cassette into the chromosome so no resistance gene remains to be transferred.
Discussion of Future Application Risks
Should our product ever move from bench to bedside, success will carry unavoidable shadows:
1. Deep colonisation drift：Daily use may allow the strain to move deeper into sinuses or the middle ear;
2. Off-target antibody binding: Nasal scFv may cross-react with respiratory mucins, altering mucociliary clearance or triggering chronic rhinitis.
3. Unauthorised re-tooling：Freeze-dried powder is room-temperature stable; unregulated labs could extract the plasmid, swap the scFv for an anti-cytokine binder, and still label it “probiotic”.
4. Environmental shedding：Sneezing releases 10⁴–10⁵ cfu per droplet; the strain survives 48 h on phones or toys, potentially colonising non-consenting household contacts together with the ermB plasmid.
These are only the risks we already foresee; others will certainly appear. We therefore commit to a standing surveillance plan: periodic swabs of users’ nostrils, tables and phones to confirm the strain is gone within four weeks and that ermB has not jumped. If either signal turns positive, we will rinse, disinfect, report and, when necessary, recall.
References
[1]Luoto R, Ruuskanen O, Waris M, Kalliomäki M, Salminen S, Isolauri E. Prebiotic and probiotic supplementation prevents rhinovirus infections in preterm infants: a randomized, placebo-controlled trial. J Allergy Clin Immunol. 2014 Feb;133(2):405-13. doi: 10.1016/j.jaci.2013.08.020. PMID: 24131826; PMCID: PMC7112326.
[2]Spacova I, Petrova MI, Fremau A, et al. Intranasal administration of probiotic Lactobacillus rhamnosus GG prevents birch pollen-induced allergic asthma in a murine model. Allergy. 2019;74:100–110.
[3]General Biosafety Standard for laboratories for causative bacteria of the People's Republic of China, WS 233—2017.
[4]Safety Regulations for Higher Education Laboratory, Department of Education and Science [2023] No. 5.
[5]Laboratories - General requirements for biosafety, GB 19489-2008.
[6]Regulation on the Bio-safety Management of Pathogenic Microbe Labs.
[7]General Guidelines for Biosafety in Microbiology and Biomedical Laboratories, WS 233-2002.
[8]Regulations on the Administration of Medical Wastes (2011 Revision).
[9]Ministry of Health. Implementation Measures for the Management of Medical Waste of Medical and Health Institutions. 15 October 2003.
[10]National Health Commission of the People's Republic of China. Medical Waste Classification Catalogue (2021 Edition). 2021.
[11]Ministry of Environmental Protection and Ministry of Health. Medical Waste Packaging, Containers and Warning Sign Standards (HJ 421-2008). 2008.
[12]United States Government Policy for Oversight of Dual Use Research of Concern and Pathogens with Enhanced Pandemic Potential. 2024.
[13]World Health Organization. “What Is Dual-Use Research of Concern?” WHO, 18 Mar. 2022, https://www.who.int/news-room/questions-and-answers/item/what-is-dual-use-research-of-concern.
[14]McInnes RS, McCallum GE, Lamberte LE, van Schaik W. Horizontal transfer of antibiotic resistance genes in the human gut microbiome. Curr Opin Microbiol. 2020 Feb;53:35-43. doi: 10.1016/j.mib.2020.02.002.