In the iGEM competition, biosafety serves as the cornerstone of all innovative practices and is the core demonstration of a team's sense of social responsibility.

Our project, "ReGenStitch," is dedicated to developing a multifunctional, absorbable, novel surgical suture designed to improve the postoperative recovery experience for patients, particularly after procedures like cesarean sections. To achieve this goal, we have established a comprehensive, multi-layered safety and security framework that spans the entire project lifecycle, from initial design to final application.

Figure 1: The Safety and Security Framework of the ReGenStitch Project

Our safety framework is structured across the following three tiers:

1.Safe Project Design: We have fundamentally mitigated the risk of releasing genetically modified organisms (GMOs) by ensuring our final product is a purified, cell-free biomaterial. We have meticulously selected a probiotic chassis organism of Risk Group 1, utilized biological parts sourced from the iGEM whitelist or safe species, and chosen final products (bacterial cellulose, chitosan oligosaccharides, curcumin) with well-documented biocompatibility.

2.Safe Laboratory Practices: All experiments were conducted in a BSL-1 laboratory that complies with national standards. All team members received rigorous biosafety training, passed assessments, and strictly adhered to Standard Operating Procedures (SOPs) to ensure the safety of personnel, experiments, and the environment.

3.Safe Product Application: We have thoroughly considered the regulatory requirements for our product as a medical device to ensure its safety in clinical applications. Our aim is to provide patients with a safer and more effective solution for postoperative recovery.

Our core safety philosophy is "control at the source, safety by design." Through meticulous design, we have ensured that potential risks at every stage of our project are minimized.

2.1 Design Philosophy: An Inherently Safe Synthetic Biology Product

The greatest safety advantage of our project is that the final deliverable—the ReGenStitch suture—is a lifeless, cell-free biomaterial, not a living genetically modified organism. All functional components (bacterial cellulose, chitosan/oligosaccharides, curcumin) are produced by engineered microbes and are then isolated and purified. This strategy fundamentally eliminates the risk of releasing GMOs into the environment and precludes the possibility of in vivo colonization or unintended immune responses in patients. This serves as the first and most robust line of defense in our project's biosafety strategy.

2.2 Safe Chassis Organisms

Based on our different production needs, we selected chassis microorganisms that are all classified as Biosafety Level 1 (BSL-1), posing no threat to healthy adults or the environment.

·Escherichia coli Nissle 1917 (EcN): As the primary chassis for producing bacterial cellulose and curcumin, EcN is a probiotic with a century-long history of clinical use. Mutaflor®, a therapeutic for IBD with EcN as its main component, is marketed in Europe and widely used to treat intestinal disorders. Its genome contains no known virulence factors or pathogenicity islands, and its safety has been thoroughly validated. Selecting EcN as the production chassis for a medical-grade material allows us to prevent contamination from harmful impurities like endotoxins from the very beginning, ensuring the high biological safety of our products.

Figure 2: Mutaflor®, a therapeutic for IBD with EcN as its main component

·Other Ancillary Strains: The Bacillus subtilis and Acetobacter sp. strains used in the chitosan pretreatment phase are Generally Recognized as Safe (GRAS) organisms, widely used in the food fermentation industry. In our project, they serve only as ancillary strains and have not undergone any genetic engineering, posing no harm to humans or the environment.

2.3 Safe Parts

All core genetic components used in our project are derived from safe, non-toxic species. The proteins they encode have well-defined functions and are not involved in the synthesis of any toxins or pathogenic factors.

Bacterial Cellulose (BC) Synthesis System:bcsA and bcsB genes: These genes together form the cellulose synthase enzyme. Their function is to catalyze the synthesis of cellulose from UDP-glucose, which is a natural, non-toxic biopolysaccharide. The working process of this system generates no harmful byproducts.

Chitosan Oligosaccharide (COS) Production System:CHI-1 gene: This gene encodes chitosanase, which is itself non-toxic. Its catalytic product, COS, is derived from the enzymatic or acid hydrolysis of chitosan, a natural process. We have immobilized this enzyme on the cell surface using surface display technology, which facilitates subsequent recovery and purification and prevents the free release of the enzyme. The working process of this system requires no other additives.

Curcumin Synthesis System:4CL1, DCS, CURS1 genes: These genes originate from common plants such as Arabidopsis thaliana and turmeric. They collectively catalyze the production of curcumin. The catalytic process generates no harmful byproducts, and its safety has been validated in the laboratory.

2.4 Safe Materials and Products

All components of the ReGenStitch suture are materials of natural origin with good biocompatibility and biodegradability.

Figure 3: The components of ReGenStitch, their origins, and functions

Bacterial Cellulose (BC): As the backbone of the suture, BC has excellent biocompatibility and has been widely used in the medical field as wound dressings and tissue engineering scaffolds, without causing immune rejection in the human body.

Chitosan & Chitosan Oligosaccharides (COS): These substances are derived from natural waste materials like shrimp and crab shells and are recognized as safe biomaterials. They possess antimicrobial and pro-healing properties and can be degraded in the body by enzymes into harmless glucosamine. COS has extremely low toxicity. Compared to chitosan, it has a lower chance of causing allergic reactions and has good water solubility, allowing it to be easily absorbed or excreted by the body. It is widely used in food, cosmetics, and medical fields.

Curcumin: Curcumin is the main active ingredient in curry and has a history of dietary use spanning thousands of years. Its safety has been confirmed through long-term dietary application and numerous modern scientific studies.

2.5 Biosecurity Considerations

Biosecurity aims to prevent the malicious misuse of biotechnology and its materials. As our project is intended for the development of a clinical medical product, we have instituted the following security measures:

Access Control: We strictly limit laboratory access to non-team members, ensuring that strains, plasmids, and critical data are only accessible to authorized personnel.

Data Security: All experimental data and genetic sequences are stored on password-protected cloud and local servers to prevent unauthorized access and duplication.

Responsible Conduct Education: We have provided all team members with education on biosecurity and the dual-use potential of research, ensuring that everyone understands and adheres to the principles of responsible scientific conduct.

III. Safe Laboratory Practices

We are deeply aware that rigorous experimental procedures are fundamental to ensuring the safety of our personnel and the environment. All of our team's experimental activities were conducted in a safe and regulated environment.

Figure 4: Laboratory Safety Scenarios. A: Safe laboratory operations; B: Waste segregation and disposal; C: Safe centrifuge operation.

3.1 Laboratory Certification and General Guidelines

Figure 5: Laboratory Attire Requirements (BSL-1)

·Laboratory Level: Our experimental work was carried out in a certified Biosafety Level 1 (BSL-1) laboratory, and we adhered to enhanced BSL-1 management protocols. The laboratory is equipped with first-aid supplies, and we ensure that fire exits are unobstructed and fire safety equipment is functional.

·Safety Training and Assessment: All members entering the laboratory were required to participate in systematic safety training organized by our institution. The training covered topics such as microbiological handling, chemical management, and emergency procedures. Only after passing a written examination were members granted access.

·Personal Protective Equipment (PPE): Within the experimental area, all personnel were required to wear lab coats, disposable nitrile gloves, and safety goggles at all times. When handling specific chemicals or performing operations that could generate aerosols, additional protection such as face masks was used, or the work was conducted within a biological safety cabinet or fume hood.

3.2 Safe Experimental Operations

• Aseptic Technique: The laboratory bench was disinfected with 70% ethanol before and after use. All operations were performed near the flame of an alcohol lamp to maintain a localized sterile environment, preventing contamination from external microbes and the release of engineered bacteria.
• Instrument and Equipment Safety: Centrifuges were strictly balanced before use. High-temperature and high-pressure equipment, such as autoclaves, were operated only by specially trained personnel. Electrical operations, like electrophoresis, were conducted ensuring there was no risk of electrical leakage. Sonicators were operated in an ice bath to prevent overheating, which could cause reagent volatilization or container rupture. Standard Operating Procedures (SOPs) for all equipment were posted nearby.
• Chemical Management: All chemical reagents were clearly labeled and stored in designated chemical cabinets according to their properties (e.g., acids, bases, flammables). Operations involving corrosive or volatile reagents were performed inside a fume hood while wearing acid- and alkali-resistant gloves.

3.3 Waste Segregation and Disposal

We established a strict waste disposal protocol to ensure that all experimental waste was managed safely, thereby preventing environmental contamination.

Table 1: Waste Disposal Methods

Waste Type	Description	Disposal Method
Infectious Solid Waste	Petri dishes, centrifuge tubes, pipette tips, etc., containing engineered bacteria.	Placed in yellow biohazard bags, autoclaved at 121°C for 20 minutes, and then disposed of as medical waste by a professional company.
Infectious Liquid Waste	Waste bacterial cultures, media, etc.	Soaked overnight in a chemical disinfectant such as 84 Disinfectant, or directly autoclaved. After confirming inactivation, treated as general laboratory wastewater.
Chemical Liquid Waste	Strong acids, strong bases, organic solvents, etc.	Collected separately in designated corrosion-resistant waste containers, labeled with contents, and collected by the university's environmental protection department for unified treatment.
Glass and Sharps	Broken glassware, scalpel blades, etc.	Placed in designated yellow sharps containers. Once full, the containers are sealed and disposed of as medical waste.
General Waste	Waste paper, clean packaging materials, etc.	Disposed of in regular trash bins.

As a medical device intended for human use, the safety of ReGenStitch is our paramount consideration.

4.1 Target Population and Application Scenarios

Our product is primarily intended for patients undergoing cesarean sections and other surgical procedures requiring skin or soft tissue closure. Post-cesarean wounds are large, have a long recovery period, and the mothers are in a special physiological and immunological state, placing higher demands on the biocompatibility, anti-infection capabilities, and healing-promoting properties of sutures. The design of ReGenStitch is precisely tailored to meet these specific needs. Through its multifunctional properties, it aims to reduce the risk of postoperative complications such as infection, pain, and hypertrophic scarring, and to avoid the discomfort and inconvenience of secondary suture removal.

4.2 Real-World Production and Development Pathway

We envision that the journey of ReGenStitch from the laboratory to clinical application will follow a rigorous medical device development pathway:

1. Scale-up Production: In a facility compliant with Good Manufacturing Practices (GMP), high-purity bacterial cellulose, chitosan oligosaccharides, and curcumin will be obtained through large-scale fermentation of our engineered strains.
2. Suture Fabrication: The three core raw materials will be mixed with other medical-grade excipients in precise ratios. The mixture will then be processed into suture filaments via wet spinning, stretching, and drying, followed by braiding, coating, and sterilization.
3. Quality Control and Compliance: The product will need to pass a series of stringent preclinical tests, including biocompatibility testing, mechanical property testing, in vitro antimicrobial/anti-inflammatory efficacy validation, and in vivo degradation and tissue response studies in animals.
4. Clinical Trials and Approval: After obtaining approval from an ethics committee, clinical trials will be conducted to verify its safety and efficacy in real surgical scenarios. Finally, a registration application will be submitted to regulatory bodies such as the National Medical Products Administration (NMPA). The product can only be launched after receiving approval.

Through this series of rigorous development and validation processes, we will ensure that ReGenStitch not only embodies an innovative concept but also possesses safe and reliable performance, capable of genuinely solving the series of problems patients encounter during postoperative recovery.

Our project began with the goal of protecting vulnerable patients during their difficult postoperative recovery; therefore, product safety has been a key focus throughout our entire project. Through an inherently safe product design, strict and standardized laboratory practices, and forward-looking considerations for future applications, we have built a solid safety foundation for the "ReGenStitch" project. This responsible approach to research reflects the iGEM competition's mission to promote the healthy development of synthetic biology for the tangible benefit of humanity. Through this project, we are committed to bringing an innovative, efficient, and unequivocally safe product from concept to reality.

1. https://www.nmpa.gov.cn/
2. https://openstd.samr.gov.cn/
3. CDC / NIH《Biosafety in Microbiological and Biomedical Laboratories》
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