Safety and risk prevention form the prerequisite for project execution, to which our team consistently attaches paramount importance. Guided by the iGEM ethos, we are dedicated to developing an efficient, convenient, and cost-effective method for screening folate metabolism gene abnormalities. To ensure safety and controllability throughout all experimental stages, we have implemented a series of stringent management measures to minimise potential hazards to team members, other laboratory personnel, and society at large.

Our team rigorously adheres to iGEM safety standards. Building upon a solid foundation of fundamental laboratory skills, we proactively participate in diverse safety training programmes. Recognising the critical importance of safety safeguards for this project— which involves techniques such as qPCR, RPA, and agarose gel electrophoresis— we have not only pre-emptively identified potential risks and formulated mitigation strategies but also continuously conducted safety self-assessments and optimisations throughout the project's progression. Notably, we have implemented corresponding measures to address potential project risks, thereby establishing a robust safety foundation to secure broader societal recognition for our outcomes.

In summary, we strictly adhere to national and institutional laboratory safety policies, regulating all project operations to the highest laboratory safety standards. While ensuring the safety of our own project, we shall also exert every effort to contribute to the secure development of synthetic biology and the safe application of related products.

Experiments were conducted in the laboratories of the School of Food and Pharmaceutical Engineering at Nanjing Normal University. Our laboratories are classified as Grade 1 Standard Microbiology Laboratories, compliant with the Regulations on the Administration of Biosafety of Pathogenic Microorganisms of the People's Republic of ChinaManagement of Pathogenic Microorganisms (https://www.gov.cn/gongbao/content/ 2019/content_5468882.htm) and the Standard for Laboratory Safety in Institutions of Higher Education(https://www.gov.cn/zhengce/zhengceku/2023-02/21/5742498/files/493d9b7d76014c50903cbc16e09c2253.pdf).

All our laboratory practices comply with the Nanjing Normal University Laboratory Safety Management Regulations (ssc.njnu.edu.cn/info/1024/1629.html). The laboratory features clearly defined and orderly functional zones to ensure operational efficacy. iGEM members receive guidance and supervision from laboratory specialists when using equipment to prevent accidents. Safety labels and precautions are displayed beside potentially hazardous apparatus, such as centrifuges, autoclaves, and gel electrophoresis units, where improper operation or other factors may pose risks. Users must log instrument usage in dedicated records, specifying operating times and personnel.In addition, apart from essential safety supplies such as goggles, masks, protective gloves, and lab coats, we are also equipped with emergency equipment including first-aid kits, emergency showers, and eye wash stations, which fully ensures the safety of laboratory personnel.

(1) Flame-retardant and waterproof workbenches: These can be used for experiments with low aseptic operation requirements or for placing various instruments. They are resistant to a certain degree of heat, organic solvents, acid-base reagents, and other chemicals.

(2) Mechanical ventilation systems: All systems have been treated to be windproof, rainproof, and debris-proof, and the exhaust systems are equipped with filters.

(3) Chemical fume hoods: These are used for the addition and preparation of toxic, volatile, or fine-particle chemical reagents and drugs.

(4) Laminar flow cabinets and biosafety cabinets: These facilitate a series of aseptic operation experiments.

(5) Autoclaves and other sterilization equipment;

(6) Emergency showers and eye wash stations;

(7) First-aid kits;

(8) Emergency lighting devices;

(9) Smoke alarms and fire extinguishers.

Personal protection is the most fundamental step to ensure laboratory safety, as it can prevent harm to laboratory personnel from most accidents. Therefore, prior to the project initiation, we provided all team members with comprehensive laboratory safety and skill training, ensuring that each member has a thorough understanding of the laboratory safety management system and the correct and standardized operation of various instruments in the laboratory. During our experiments, there is at least one PI or Instructor present to ensure project safety and provide timely guidance.

All team members have a basic foundation in experimental skills. They have systematically studied and passed the course Introduction to Safety Engineering, gaining sufficient understanding of the operational risks in the laboratory environment and familiarity with the procedures for mitigating and addressing potential accidents. In addition, we have arranged pre-drills on experimental protection for each team member to ensure the correctness and effectiveness of protective work.

Before the project started, Professor Li Bingzhi conducted another safety training session tailored to this project. The training included a comprehensive introduction to the laboratory's rules and regulations, laboratory safety management standards, and methods for preventing and addressing hazardous situations. It also covered detailed explanations of the standard operation procedures for laboratory instruments, as well as the storage and usage methods of various chemicals. Furthermore, demonstrations were provided on the classification and disposal of various laboratory wastes. Finally, laboratory professionals conducted training on experimental skills and related operational procedures, such as PCR, qPCR, and gel electrophoresis.

By implementing these training measures, we have maximized the safety and reliability of the experiments, thereby reducing the potential risks of the project to team members and the external environment.

(1) Timely communication and discussion of experimental plans prior to conducting experiments help identify potential accidents in advance and reduce the likelihood of their occurrence. Effective communication also ensures that other laboratory personnel are aware of the ongoing experiments, enabling them to respond quickly and handle incidents properly if an accident occurs.

(2) Establishing and adhering to standard operating procedures (SOPs) for all laboratory activities ensures consistency and minimizes the potential for errors. These SOPs should cover everything from material handling and disposal to emergency response protocols.

(3) Conducting regular risk assessments helps identify potential hazards and vulnerabilities. This proactive approach allows for the implementation of mitigation strategies before accidents occur.

(4) Ongoing training, including refresher courses and updates on new procedures or technologies, helps maintain a high level of safety awareness.

Laboratory waste is categorized into solid waste and liquid waste.Solid waste is further divided into waste packaging, gloves/masks, and experimental waste, which are collected separately by category.Liquid waste is classified into organic waste liquid, inorganic waste liquid, and fermentation broth. Each type is collected in separate containers with clear labels, and a leak-proof tray is placed under each waste liquid bucket.There is a dedicated storage area for laboratory waste, where warning tapes are affixed on the ground and relevant signs (e.g., "Hazardous Waste") are posted on the wall. Every week, after filling out the waste disposal form, we make an appointment with a professional laboratory waste disposal company, who will send specialists to collect the laboratory waste.Especially, our experiment involves blood samples. After the experiment, we will package the blood samples in dedicated containers, which will then be delivered by designated personnel to a specified disinfection site for centralized handling. Finally, the tested blood specimens and wastes will be disposed of by a specialized agency using incineration.

Buffers used in experiments, such as TBE buffer and red blood cell lysis buffer, require pH adjustment, so we need to use hydrochloric acid, a chemical controlled by the state. We strictly adhere to the application procedure for hazardous chemicals:First, an application report is filled out, detailing information including the applicant, application date, name of the hazardous chemical, dosage, and intended use, which is then signed by the applicant’s supervisor. The report is subsequently submitted to the hazardous chemicals management teacher for review.Upon approval, two custodians use two different keys to open the two layers of locks on the cabinet dedicated to storing hazardous chemicals. Hydrochloric acid is got under their supervision. After usage, when hydrochloric acid is returned to the warehouse, detailed information about the collection is recorded in a dedicated ledger.

The Native PAGE solution used in experiments can enter the human body of laboratory personnel through skin contact, inhalation, or accidental ingestion, potentially causing acute or chronic poisoning. During experimental operations:All students wear full sets of protective equipment.Volatile toxic chemicals are handled inside a fume hood to avoid direct pouring or heating on open workbenches.We follow the "minimum dosage principle" to minimize the use of toxic chemicals, thereby avoiding waste and safety hazards.

The fluorescent probe designed in our laboratory is a 16-nt single-stranded DNA. Its 5’ end is modified with a fluorescent group, the 3’end with a quencher group, and an RNA base is modified in the middle. This fragment does not have the ability to self-replicate in the natural environment.In addition, the chassis cell used in the experiment is Escherichia coli BL21 (DE3), which is classified as Biosafety Level 1 (BSL-1) and has low pathogenicity to humans and animals. Moreover, during bacteria-related experiments, we conduct strict sterilization before and after the experiment, as well as regular disinfection and environmental testing. We are capable of responding to various emergency situations and providing comprehensive, multi-level protection.

Blood samples may carry blood-borne pathogens (e.g., HBV, HCV, HDV, HTLV-I/II, Plasmodium, etc.). Improper handling of samples may expose laboratory personnel to infection risks. Cross-contamination between blood samples may also lead to erroneous test results, thereby affecting the experiment.In this project, all blood samples used are provided by qualified cooperative hospitals, fundamentally eliminating the possibility of infectious pathogens (such as bacteria or viruses) in the samples. In addition, all subjects signed an informed consent form before providing blood samples, and the hospital did not attach any personal information of the subjects when providing the blood samples, thus eliminating the risk of information leakage at the source.

During the project operation, we strictly adhere to laboratory operating standards and have also taken the following measures to ensure safety:

(1) Personal protection: Wearing full sets of protective equipment and receiving safety training in advance;

(2) Process specification: Standardizing sample handling procedures to reduce exposure risks, and strictly controlling handling time to ensure sample stability;

(3) Environmental management: Effectively reducing the risk of cross-contamination of blood samples through workbench partitioning and staggered experiment scheduling.

At the end of the project, the POCT kit we designed has the potential to become a fast and simple home-based detection method where both the experimental reaction and result output are conducted in a sealed container, reducing costs and contamination while improving the convenience of detection.

RPA is the abbreviation of Recombinase Polymerase Amplification, an isothermal nucleic acid amplification technology. It uses recombinase to bind with specific primers and form a dynamic complex. With the synergistic effect of single-stranded DNA-binding proteins, the complex unwinds the single-stranded template and prevents it from reannealing, thereby replacing the high-temperature denaturation step in traditional PCR. Subsequently, DNA polymerase with strand displacement activity can continuously extend the primers under a constant temperature of 37-42°C, enabling exponential amplification of nucleic acids.

The application of RPA technology offers the following advantages:

(1) It does not require sophisticated equipment such as thermal cyclers, allowing for isothermal in vitro amplification;

(2) It integrates the advantages of rapidity, sensitivity, and portability, which aligns with the R&D goal of rapid detection test strips expected in this project;

(3) It features simple equipment, easy operation, and low operating costs;

(4) The technology itself causes no pollution or harm to the environment.

Our product is ultimately presented in the form of test strips, assisting medical institutions in conducting efficient, convenient, and cost-effective screening for folate metabolism disorders. Both the test strips and their outer casings are safe and environmentally friendly, and the safety of the usage scenarios ensures the safety of product recycling and disposal. Blood samples involved in the detection process will be temporarily stored uniformly by medical institutions and subjected to harmless treatment.

To ensure data security and privacy protection, we safeguard the safety of blood samples throughout the entire process. Experimental data strictly complies with the confidentiality system and is only processed and analyzed by internal personnel, minimizing the risk of personal information leakage. All team members have received professional ethics training; all experimental data has undergone scientific and reasonable interpretation, and is solely used for research purposes without infringing on personal rights and interests.

To minimize ethical and social risks, we proactively identified potential ethical dimensions involved and made corresponding adjustments. When processing experimental data, we avoid absolute interpretation of results and strictly prohibit the use of labeling expressions such as "pathogenic gene carriers" and "populations with genetic defects". The final genetic testing results are only used for medical decision-making, not as a basis for social evaluation, and the extension of the results' social attributes is strictly restricted. We have passed the biomedical ethics review and obtained the Biomedical Research Ethics Review Form from NNU (Ethics Review No.: NNU202506013).

Before the project initiation, team members carefully studied and reviewed the following documents and policies to ensure that the project content complies with relevant policies and regulations:
(1) National Basic Public Health Service Specification (Third Edition)(http://jkw.lasa.gov.cn/wswyh/fwgk/202206/9ad7f743ca124da89685486098a2793f.shtml )
(2) Plan for Enhancing Birth Defects Prevention and Control Capabilities (2023-2027)(https://www.nhc.gov.cn/fys/c100078/202308/8230a24d8c8846a9bad6545d7b229162.shtml)
(3) Measures for the Administration of Prenatal Diagnostic Technologies(https://www.nhc.gov.cn/wjw/c100221/202201/681c3d285fe149abb8fcf2779f6aef21.shtml)