In the iGEM competition, fostering a robust culture of safety and responsible innovation is of paramount importance. Embracing this core principle, our team has integrated the ProBabyotics project with a comprehensive biosafety framework into every facet of our work, from initial design to public engagement. This document outlines our multi-layered approach to safety. Our strategy begins with a safe-by-design approach, ensuring the project's inherent safety by selecting the probiotic Escherichia coli Nissle 1917 (EcN) as our chassis—a strain with a century-long history of safe clinical use. The core products we aim to produce, including 2'-FL, β-galactosidase, and trypsin, all have well-documented safety profiles widely certified by global regulatory agencies. In the laboratory, all experiments are conducted under strict BSL-1 standards, supported by rigorous protocols for chemical and biohazardous waste management. To guarantee personnel competence, we implemented a unique "Three-Tier Safety Qualification System," which requires every member to pass theoretical training, a written examination, and a practical skills assessment before working independently. Finally, our commitment extends to safe human practices and education. We actively raise awareness and disseminate knowledge to the broader community by creating educational lab safety posters and delivering presentations on historical lab accident case studies, reinforcing the shared responsibility of maintaining a safe research environment.

In the ProBabyotics project, we firmly believe that responsible innovation is the only path toward meaningful scientific progress. For us, biosafety is not a checklist to be completed but a core culture integrated into our team's mindset and daily work. We are committed to conducting proactive risk assessment and management at every stage of our project—from conceptual design and genetic engineering to experimental execution, waste disposal, and even the application scenarios of our final product. Our goal is not only to ensure the safety of our team members and the environment but also to contribute our understanding and practices of safety to the iGEM community, ensuring that the power of synthetic biology is used to benefit humanity.

Figure 1：Biosafety Philosophy

A safe and reliable project begins with its design. We have conducted careful safety assessments for every aspect of our project, from the chassis organism and functional components to the production process and proposed implementation.

Safe Chassis

Our choice of E. coli Nissle 1917 (EcN) as the project's chassis organism is the cornerstone of our safety strategy. This decision is based on several key safety advantages:

Extensive History of Clinical Validation: As a probiotic, EcN has a history of safe use spanning over a century since its discovery in 1917. It has been widely used to treat various intestinal disorders, and its safety has been confirmed in numerous clinical studies (Sturm et al., 2024).

Recognized as Non-Pathogenic: Unlike many other E. coli strains, the EcN genome does not contain genes encoding common virulence factors such as Shiga toxins or hemolysins. Its safety as a production chassis is widely acknowledged (Sun et al., 2023; Pantoja-Angles, 2025).

Low-Immunogenicity Lipopolysaccharide (LPS): EcN possesses a unique LPS structure, which exhibits significantly lower immunogenicity compared to pathogenic Gram-negative bacteria and does not trigger a strong inflammatory response. This feature is critical for a project aimed at producing ingredients for infant food, as it greatly reduces safety risks during the final product purification process (Sturm et al., 2024).

Well-Defined Genetic Background: The complete genome sequence of EcN has been published, providing a clear genetic background. This facilitates precise and controllable genetic engineering, thereby preventing unforeseen genetic variations (Sun et al., 2023).

By selecting EcN as our chassis, we ensure that our "cell factory" is itself a time-tested and human-friendly safety platform (Creative Biolabs, n.d.).

Figure 2：Safety features of E. coli Nissle 1917

Safe Parts and Products

The safety of the three core products produced by our engineered bacteria is well-documented:

2'-Fucosyllactose (2'-FL)

2'-FL is one of the most abundant oligosaccharides in human milk and a core functional component. Its safety has been widely recognized by global regulatory agencies. While the U.S. Food and Drug Administration (FDA) has long recognized 2'-FL produced in E. coli strains as "Generally Recognized as Safe" (GRAS) (e.g., GRN No. 749, 897, 929), permitting its use in infant formula (FDA, 2019; FDA, 2020), China only recently approved the use of 2'-FL in infant foods in 2024. This regulatory change has paved the way for the incorporation of 2'-FL into infant formula in China, and our project aligns with this new regulation to introduce 2'-FL as an ingredient in infant formula. Studies have shown that the addition of 1 g/L of 2′-FL in infant formula is safe (Puccio et al., 2017).

β-Galactosidase

β-Galactosidase, commonly known as lactase, is the enzyme essential for digesting lactose in the human body. It has been safely used as a food additive worldwide for decades. In 2022, the European Food Safety Authority (EFSA) evaluated β-galactosidase derived from Aspergillus oryzae and concluded that no adverse effects were observed at any tested dose, establishing its "No Observed Adverse Effect Level" (NOAEL) (EFSA Panel on Food Contact Materials, Enzymes and Processing Aids, 2022).

Trypsin

Trypsin is a key digestive enzyme, naturally found in the pancreas of pigs, responsible for breaking down proteins in the small intestine. As an exogenous enzyme, it is derived from pigs and is highly biocompatible, with no known immunogenic or toxic risks when used appropriately. In our project, we aim to produce an enzyme identical in sequence and structure to porcine trypsin through genetic engineering. This ensures that the enzyme maintains the same functionality and safety profile as the natural version, making it suitable for use in digestion-related applications.

Safety Process and Implementation

From the production of core components to formula processing and quality control, we established a comprehensive safety assurance system. 2′-FL is produced in a closed fermentation system and subsequently purified to remove impurities; enzyme preparations undergo activation and purification, with all waste treated by autoclaving; during formula processing, enzymes are applied for pretreatment—trypsin hydrolyzes casein proteins while β-galactosidase breaks down lactose—to reduce risks of allergy and intolerance in infants; throughout the process, HPLC analysis, residue detection, and microbial monitoring are conducted to ensure full compliance with both EU and Chinese national safety standards.

All our experimental activities were conducted in a Biosafety Level 1 (BSL-1) laboratory, in compliance with Chinese national standards and iGEM safety policies. We have established a comprehensive laboratory safety management system.

Laboratory Environment and Management

Our laboratory is equipped with complete safety infrastructure to address potential risks:

Safety Equipment: We are equipped with biosafety cabinets, fume hoods, emergency eyewash stations, safety showers, and standard-compliant fire extinguishers. All equipment is regularly maintained and inspected.

Chemical and Waste Management: All chemicals are stored in explosion-proof cabinets with complete Safety Data Sheets (SDS) readily available. All biohazardous waste (e.g., culture plates and liquids containing engineered bacteria) is sterilized by autoclaving before being collected by a professional medical waste disposal company, ensuring no harm to the environment.

Figure 3：Photo of laboratory

Personnel Training and Competence

We believe the most effective safety measure is a well-trained researcher. To this end, we designed and implemented a "Three-Tier Safety Qualification System":

Tier 1: Theoretical Knowledge Training: Before entering the lab, all team members must attend systematic safety training conducted by a senior researcher. The training covers general lab guidelines, chemical safety, biosafety regulations and emergency response protocols.

Figure 4：Photo of Lab Safety Manual

Tier 2: Written Examination: Members must complete a comprehensive safety knowledge exam covering all key points from the training. A score of 90% or higher is required to proceed to the next tier.

Figure 5：The written examination is a crucial step in our safety qualification system, ensuring every member has mastered essential safety knowledge.

Tier 3: Practical Skills Assessment: Under the supervision of a mentor, members must demonstrate proficiency in key operations (e.g., aseptic technique, pipette usage, waste disposal) and simulate responses to emergencies (e.g., a chemical spill). Only after passing all three tiers are members granted authorization to conduct experiments independently.

Figure 6：Before entering the clean bench to start the experiment, the experimenter (team member) will wear gloves and be sprayed with alcohol for disinfection.

Figure 7：Three-Tier Safety Qualification System

We believe that biosafety education is a responsibility of every iGEM team. We not only adhere to strict internal standards but are also committed to disseminating safety knowledge to the broader community.

Laboratory Safety Educational Posters

We designed a series of educational posters on laboratory safety. we aim to remind researchers to remain vigilant at all times. These posters are displayed on the bulletin boards of biology and chemistry laboratories at our university.

Figure 8：Laboratory Safety Educational Posters

Laboratory Safety Presentations

We delivered a series of PPT presentations focusing on historical laboratory accident cases. By reviewing real incidents that occurred in different universities, we highlighted the consequences of neglecting safety rules and demonstrated the importance of preventive measures. Through these presentations, participants gained a deeper understanding of laboratory safety, strengthened their awareness of potential risks, and were reminded of the shared responsibility to maintain a safe research environment.

Figure 9：Laboratory Safety Presentations

1. BOC Sciences. (2023, June 14). Fermentation and Purification Update: Endotoxin and Its Removal. Retrieved from https://www.bocsci.com/blog/fermentation-and-purification-update-endotoxin-and-its-removal/
2. Creative Biolabs. (n.d.). E. coli Nissle 1917. Retrieved September 5, 2025, from https://live-biotherapeutic.creative-biolabs.com/escherichia-coli-nissle-1917-482.htm
3. Creative Biogene. (n.d.). Downstream Processing Techniques. Retrieved September 5, 2025, from https://microbiosci.creative-biogene.com/downstream-processing-techniques.html
4. EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (EFSA CEF Panel), Barat Baviera, J. M., Bolognesi, C., Chesson, A., Cocconcelli, P. S., Crebelli, R., ... & Zorn, H. (2022). Safety evaluation of the food enzyme β-galactosidase from the non-genetically modified Aspergillus oryzae strain GL 470. EFSA Journal, 20(10), e07572. https://www.efsa.europa.eu/en/efsajournal/pub/7572
5. Food and Drug Administration (FDA). (2019). GRAS Notice GRN 000749: 2'-O-fucosyllactose. Retrieved from https://www.fda.gov/files/food/published/GRAS-Notice-GRN-749-Part-1.pdf
6. Food and Drug Administration (FDA). (2020). GRAS Notice 897, 2'-O-fucosyllactose. Retrieved from https://www.fda.gov/media/136755/download
7. Pantoja-Angles, A. (2025, August 31). Engineering E. coli Nissle as safe chassis for delivery of therapeutic peptides. bioRxiv.http://www.biorxiv.org/content/10.1101/2025.08.30.673081
8. Sturm, A., Rilling, K., Baumgart, D. C., & Gysling, E. (2024). Emerging strategies for engineering E. coli Nissle 1917-based therapeutics. Trends in Biotechnology.
9. Sun, D., et al. (2023). Engineering E. coli Nissle 1917 as a microbial chassis for therapeutic and industrial applications. Biotechnology Advances, 67, 108202.
10. Puccio, G., et al. (2017). Effects of infant formula with human milk oligosaccharides on growth and morbidity: a randomized multicenter trial. Journal of pediatric gastroenterology and nutrition, 64(4), 624-631.