Safety

General Safety Rules

• We tied back long hair to minimize exposure to chemicals.
• Protective gloves, closed-toe shoes, and lab coats (and other personal protective equipment) were worn to safeguard against exposure to chemicals.
• Foods and drinks were never ingested inside the laboratory.
• We discarded contaminated materials immediately after use.
• All equipment was handled with care to prevent breakage and injury.
• Nobody worked alone in the lab.
• Every lab member behaved responsibly without any horseplay.
• Work surfaces were kept clean and uncluttered to avoid accidents.
• Nobody wore loose clothing or dangling jewelry
• We confirmed that the laboratory was free from contamination after each session.

Specific Policy Compliance

• “Prohibited Activities” Policy: We did not use organisms from Risk Group 3 or 4; release or deploy a genetically modified organism outside the lab; test our product on humans (including ourselves); or use parts from an organism in Risk Group 4.
• “White List” Policy: Our wet lab work did not involve any Risk Group 3 or 4 organisms, non-Whitelisted organisms, or the SARS-CoV-2 virus.
• “Release Beyond Contaminant” Policy: We did not release or use any parts of the genetically modified organisms outside the laboratory.
• “No Human Experimentation” Policy: Our project did not involve any human experimentation. Our procedure never used human samples, nor did it have any direct contact with our lab members and engineered organisms. Furthermore, our project did not involve any human subjects research, including surveys, interviews, and public engagement during wet lab sessions.
• “Animal Use” Policy: The project did not involve the use of animals in any part of our experiments. No animals or animal samples were used.
• “Antimicrobial Resistance” Policy: No antimicrobial resistance factors were created or enhanced in any organism during our experiments.
• “Gene Drives” Policy: Our wet lab work did not include the use of gene drives.
• “Environmental Samples” Policy: We did not isolate organisms from outside samples and ensured that any non-traditional materials were used only within the guidelines.

About Our Lab

(Figures 1~2: Photo of our lab with relevant safety features.)

Our project involved lab work under Biosafety Level 2 (moderate containment). We utilized an open bench and a biosafety cabinet to handle biological materials.

About Our Project

On Our Project: Our 2025 iGEM Korea-HS project aims to develop a bio-upcycling platform that transforms fruit peel waste into valuable products using an engineered probiotic bacterium. Specifically, we are engineering Lactobacillus reuteri to express a codon-optimized polygalacturonase (PGXc) gene derived from Aspergillus niger. This enzyme enables L. reuteri to efficiently degrade pectin, a major polysaccharide found in citrus and other fruit peels. Our long-term goal is to reduce food waste accumulation and valorize agricultural byproducts by converting fruit peels into useful biomaterials such as bioethanol, bioplastics, or prebiotic oligosaccharides. This project demonstrates a sustainable synthetic biology solution to address global food waste challenges and promote circular bioeconomy principles.

• We used Lactobacillus reuteri ATCC 55730 as a whole organism.
• Our project only used genetic parts that are already in the registry.
• None of our parts are hazardous on their own/or in the context of our project.
• We ordered our DNA/RNA from a company, a member of the IGSC.
• We did not use any kind of hazardous chemicals in our project.
• We did not use an Artificial Intelligence tool(s) for the project’s development.

On Our Parts: While our project involves engineering Lactobacillus reuteri ATCC 55730, a GRAS (Generally Recognized As Safe) probiotic strain, and using a codon-optimized pgxC gene from Aspergillus niger (a non-pathogenic filamentous fungus), our risk assessment indicates no significant biosafety risks. The engineered organism is contained within a biosafety level 2 (BSL-2) laboratory, and all work is conducted following strict biosafety protocols, including proper use of personal protective equipment (PPE), sterilization of waste via autoclaving, and supervision by trained staff. The plasmid design is non-conjugative, minimizing any risk of horizontal gene transfer. While the DNS assay involves chemicals such as 3,5-dinitrosalicylic acid, standard chemical handling procedures, including use in a fume hood and proper PPE, effectively mitigate any potential hazards. Overall, with these containment measures and safe laboratory practices in place, our project presents no significant safety or environmental risks.

Materials That Pose Potential Risks

• Escherichia coli was used to clone the vectors containing the pgxC gene.
• The cloned vectors were injected into Aspergillus niger for transformation.
• Antibiotics such as Erythromycin were used for the positive selection of E. Coli.
• Coomassie Brilliant Blue R-250 was used for SDS-PAGE.
• Organic fruits were used for experimentation in evaluating the efficiency of degradation.

Managing Future Risks upon Implementation

Our project aims to develop a biological solution for the sustainable management of fruit peel waste, a significant component of global food waste. In the real world, the engineered Lactobacillus reuteri ATCC 55730 strain, capable of secreting polygalacturonase (PGXc), could be used in industrial bioreactors or local waste-processing facilities to break down pectin-rich fruit peels such as citrus, banana, and apple peels. By converting these peels into simpler sugars, the system could serve as a pre-treatment step for producing valuable bio-based products such as bioethanol, organic acids, or prebiotic oligosaccharides. The technology could be integrated into food processing plants, fruit markets, and urban waste management systems where large volumes of fruit waste are generated, reducing landfill burden and methane emissions.

Our project’s future experiments would not involve releasing beyond containment. Any accidental exposure to laboratory organisms or chemicals poses a threat to human safety; therefore, biological safety protocols must be implemented to prevent their spread.

Such biological safety protocols include physical barriers that block the spread of genetically modified bacteria, sterilization procedures to prevent accidental releases, the proper use of biosafety equipment, and strict adherence to laboratory access controls.

Our project is foundational, and we do not have a specific real-world application in mind. Also, future development of our project would not require release beyond containment.

Managing Bad Outcomes

Our engineered Lactobacillus reuteri ATCC 55730 strain is designed for use in controlled laboratory and industrial conditions, and multiple biological and practical barriers limit its ability to spread in the environment. First, L. reuteri is a generally regarded as safe (GRAS) probiotic strain that does not naturally persist in the environment, as it primarily inhabits the human gastrointestinal tract and requires specific nutrients and conditions found in laboratory media or specialized fermentation systems. Outside these controlled environments, the bacteria would likely struggle to survive due to competition from native microorganisms and the absence of suitable growth substrates such as MRS broth.

Second, the engineered plasmid carrying the pgxC gene is designed to function only within L. reuteri and lacks conjugation machinery, making it unable to transfer horizontally to other bacterial species in the environment. Additionally, our system relies on a constitutive ermB promoter that drives expression under specific conditions optimized in the lab; without these conditions, expression and survival would be severely limited. Lastly, we will implement strict biosafety measures, including proper waste treatment and inactivation of bacterial cultures before disposal, ensuring that the engineered strains are contained and do not enter natural ecosystems. These biological, genetic, and procedural safeguards collectively prevent the unintended spread of our engineered L. reuteri strain in the environment.

• We had a conversation about the harm to the environment, including the impact on wild plants and animals.
• We do not think that our project could lead to any of the bad outcomes considered.
• Upon development, our engineered organisms/parts cannot spread in the environment.

Our Experts

In managing potential risks, we will seek guidance from the IRIS LAB Safety Manager, who has expertise in laboratory biosafety, risk management, and compliance with standards related to synthetic biology. The Safety Manager will assist us in identifying and mitigating potential hazards, ensuring safe handling of genetically modified organisms, and guiding us in proper waste disposal and containment practices. If we encounter unexpected risks—such as issues related to the containment of our engineered Lactobacillus reuteri strain or unexpected results from pectin degradation experiments—we will consult the Safety Manager immediately for advice on corrective actions, adjustments to our protocols, and enhanced safety measures. This support system ensures that our project is conducted in compliance with biosafety standards and that we maintain a safe working environment for all team members.

Our Approach

Our project has implemented multiple layers of safety measures to effectively manage risks and minimize hazards associated with laboratory work. These measures include institutional oversight, strict compliance with biosafety standards, team-wide training, and proper waste disposal protocols. Specifically, our efforts included but were not limited to:

Oversight and Compliance: We were given instructions by our professor before the lab session, and were supervised by him during the experiments. Biosecurity measures are enforced at all times.

Training and Safety Sessions: All team members received training in laboratory safety prior to entering the lab. Training topics covered the following contents:

• Lab access and rules, such as clothing, eating, and drinking
• Responsible individuals (e.g., lab or departmental specialist or biosafety officer)
• Differences between biosafety levels
• Biosafety equipment (e.g., biosafety cabinets)
• Good microbial technique (e.g., disinfection and sterilization)
• Emergency procedures (e.g., chemical, fire, and electrical safety)
• Rules for transportation of samples between labs or shipping between institutions
• Physical biosecurity (e.g., tracking materials and access controls)
• Personnel biosecurity (e.g., watching for unusual behavior)
• Data biosecurity / cyberbiosecurity (e.g., managing database access)

Laboratory Biosafety and Biosecurity Measures:

• Accident reporting (e.g., a system to record any lab accidents)
• Personal Protective Equipment (e.g., wearing lab coats, gloves, eye protection, etc.)
• Inventory controls (e.g., tracking who has what materials and where they are)
• Physical access controls (e.g., controlling who can access your lab or storage spaces)
• Data access controls (e.g., controlling who can access computers or databases)
• Lone Worker or Out of Hours policy (e.g., procedures for working alone or at times)
• Medical surveillance (e.g., finding out if you get sick because of an organism or chemical)
• Waste management system (e.g., such as decontaminating waste)

Waste Management: General waste guidelines included the following:

• Autoclaving or disinfecting contaminated cultures before disposal.
• Cleaning and disinfecting incubators, centrifuges, refrigerators, microscopes, pipettors, and other equipment exposed to contaminated material.
• Discarding all media and reagents used with contamination.
• Properly sealing and labeling biohazard bags or containers before disposal.

Laboratory Practices and Equipment:

• Relevant biosafety apparatus was consistently used during experiments.
• Personal protective equipment (PPE), including lab coats and gloves, was provided to all team members and supervisors and was worn at all times.
• A communication plan was developed to ensure rapid reporting and coordinated responses to any safety incidents that may occur.

The risk management actions we checked above apply to our project in several important ways. First, we will adhere to good laboratory practices (GLP), including proper labeling, sterile techniques, and the use of personal protective equipment (PPE), to prevent accidental exposure to genetically modified organisms (GMOs). We will also adhere to institutional biosafety guidelines and maintain containment protocols, such as using biosafety cabinets when necessary and autoclaving biological waste before disposal, to prevent the release of engineered Lactobacillus reuteri into the environment. All team members will receive biosafety training provided by the IRIS LAB Safety Manager, ensuring they understand the risks associated with working with genetically engineered strains and how to handle them safely. Additionally, our project design incorporates biological containment strategies, including the use of a non-conjugative plasmid that limits horizontal gene transfer and the utilization of L. reuteri, a probiotic strain that does not naturally persist in the environment. Together, these actions ensure that our project is conducted safely and responsibly, minimizing risks to researchers, the public, and the environment.

Compliance with National Laws

In South Korea, our research adheres to a comprehensive framework of biosafety and biosecurity regulations designed to ensure safe and responsible scientific practices. Nationally, the Infectious Disease Control and Prevention Act and the Bioethics and Biosafety Act provide the legal foundation for managing biological risks in research laboratories. The Korea Disease Control and Prevention Agency (KDCA) oversees the enforcement of these laws, offering detailed guidelines on laboratory biosafety, biosecurity, and the handling of high-risk pathogens.

At our institution, we follow the IRIS LAB Laboratory Biosafety Manual, which outlines standard operating procedures for safely conducting experiments involving genetically modified organisms and biological materials. This manual ensures that all laboratory activities comply with both national regulations and international best practices. For more information on national biosafety guidelines, please refer to the KDCA's Biorisk Management resources.

Safety: Our Conclusion and Insights

Our team has completed biosafety training provided by the IRIS LAB Safety Manager, which covered topics such as the safe handling of Lactobacillus reuteri ATCC 55730, plasmid construction, sterile techniques, PPE usage, and emergency protocols. We also receive ongoing mentorship and oversight from the IRIS LAB Safety Manager, who acts as our primary resource for risk management questions and guidance.

Our experiments will take place in a designated Biosafety Level 2 (BSL-2) laboratory equipped with essential safety features, including biosafety cabinets (Class II) for handling bacterial cultures, autoclaves for sterilizing biological waste, and dedicated spaces for molecular cloning and culture work. All biological waste, including used cultures, pipette tips, and disposable materials, will be treated by autoclaving at 121°C for at least 15 minutes before disposal. Liquid waste will be treated with 10% bleach or 70% ethanol prior to disposal.