At iGEM AFMU-CHINA, safety remains our top priority. Since our first day together, we have been inspired by the motto of previous teams, "Safety is not expensive, it is priceless" for the Athens 2019 team. We are deeply impressed. Memoriam, we continue to maintain a strong commitment to safety. These enduring mottos reinforce the critical role of safety in the iGEM race. We carry our safety philosophy throughout the journey, incorporating bioethical considerations and safety assessments into our human practices in addition to laboratory protocols. This comprehensive approach ensures that safety guides all of our decisions and actions to make a responsible contribution to synthetic biology research.

We strictly follow the iGEM security policy, national biological safety regulations and outstanding team of the previous standards, integrate security into the SYNERGY system design, lab operation and future clinical into every link of fully identify, evaluate and reduce the potential risk. In view of the project involves slow virus transfection operation, immortalized macrophages (iBMDM) culture, hepatocellular carcinoma cell line processing features, we design a layered, scene training system, to ensure that all staff to grasp professional safety procedures.

• Basic biosafety training: Led by the team principal Investigator (PI) and the institutional biosafety officer, all members completed mandatory training on rules, including the Biosafety Law of the People's Republic of China implemented in 2021, the iGEM ban on unauthorized human related experiments, and the safety standards for recombinant nucleic acid molecules in line with NIH guidelines. The core modules cover the biological risks of lentivirus (such as the third-generation self-inactivating lentivirus/SIN-LV without replication ability), the correct operation method of level II type A2 biosafety tank.
• Specialized Skills Training: The team invited experts from the university’s Experimental Teaching Center to deliver hands-on training focused on cell culture safety. Standardized procedures were implemented for handling iBMDM, AML12 hepatocytes, Hepa1-6 hepatocellular carcinoma cells, and LLC lung cancer cells, covering aseptic techniques, inactivation of waste cells, and prevention of cross-contamination.
• High-Risk Scenario Emergency Drills: Simulated emergency drills were conducted for scenarios such as lentiviral spillage (covered with 1% sodium hypochlorite-soaked gauze for 30 minutes) and personnel exposure (15 minutes of rinsing under running water followed by disinfection with 75% ethanol), clearly defining the roles and responsibilities of instructors and researchers during emergencies.

In response to the potential future clinical translation needs of the SYNERGY system, a template for an informed consent form was developed under the guidance of Associate Professor Zhang Qiuju to ensure that future participants fully understand the research purpose, procedures, and potential risks (such as the risk of cytokine release syndrome). Additionally, personal data privacy protection measures were established, including de-identification of research data, to ensure the project adheres to the ethical principles of respect, beneficence, and justice.

The plasmids and cell lines used in the project were all sourced from compliant suppliers and adhere to iGEM’ s biological material usage guidelines. Any materials not included on iGEM’ s whitelist were registered in advance through the iGEM Safety Portal.

According to our laboratory biosafety level 2 (BSL-2) standard operation procedures, and considering the characteristics of projects using lentivirus and engineered cells, we have implemented a "design - audit - supervision" closed-loop control system.

• Protection and Isolation Equipment: Equipped with 2 Class II Type A2 biosafety cabinets (calibrated annually by a third party), all lentivirus operations and engineered cell cultures are performed here to prevent aerosol leakage. There is a dedicated room for real-time fluorescent quantitative PCR and flow cytometry instruments, equipped with UV-C overnight irradiation sterilization procedures.
• Emergency Equipment: Eye wash stations, emergency showers, fire blankets, and lentivirus spill response kits (containing 1% sodium hypochlorite solution and sterile gauze) are inspected and maintained monthly.
• Biological Waste: Culture media containing lentivirus and used cell culture plates are autoclaved at 121°C for 30 minutes before disposal; used pipette tips are placed into biohazard bags and incinerated by qualified companies.
• Chemical Waste: Reagents such as ethanol and sodium hypochlorite are sorted into designated containers and recycled by qualified waste disposal enterprises.
• Medical Waste: Contaminated personal protective equipment (PPE) like gloves and masks are disposed of in yellow medical waste bins according to local government regulations.
• Environmental Control: The laboratory monitors temperature and humidity daily to ensure a stable environment.
• Access Control: Only trained and approved personnel are allowed to enter the laboratory. The check-in log records the time, reason, and name of the supervisor.
• Lentivirus Storage: Stored in -80°C freezers under a dual-lock system (access requires signatures from both the PI and the on-duty graduate student). A "Syn-M project exclusive experimental account" is established to record details such as packaging information, titer, user, and destination for each batch of virus.
• Cell Line Management: Each cell line (such as iBMDM, Hepa1-6) is marked with passage numbers and storage dates, and special pipettes and culture mediums are used to prevent cross-contamination.
• Regular Checks and Audits: Focuses on verifying the integrity of the biosafety cabinet, the effectiveness of disinfectants, and the standardization of waste disposal.

The selection of chassis cells constitutes the first line of defense for biosafety in synthetic biology design. To this end, we conducted extensive literature reviews, expert interviews, consultations, and education and training programs to minimize potential violations and hazards to the environment and laboratory personnel during experimental procedures. Our SYNERGY system employs macrophages as the chassis cell, ensuring high biosafety from the outset. Compared with prokaryotic bacteria commonly used as chassis, eukaryotic macrophages cannot survive or proliferate independently outside the body, and their genetic material is naturally isolated from common environmental bacteria. This significantly reduces the risk of horizontal gene transfer and dissemination of engineered genetic material in the event of accidental release. The SYNERGY system is activated only in the presence of the liver-specific membrane protein SLC17A2, subsequently triggering concurrent overexpression of P65 and knockdown of SIRPα. This design inherently restricts the biological application scope of SYNERGY, ensuring its function is exerted exclusively within the in vivo environment of patients receiving therapeutic infusion. It thereby fundamentally avoids the risks of environmental leakage and genetic contamination associated with prokaryotic cell–based therapies, aligning with the proactive biosafety design principle of ‘prevention before occurrence’.

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