Safety

Safety is a critical topic in synthetic biology and an essential factor that must be considered in any work or product. The 'Work Safety Law of the People's Republic of China' clearly stipulates that our country's safety management work must adhere to the principle of 'safety first, prevention foremost, and comprehensive management.' In all production processes, protecting human life must be the top priority. Our project aims to create a controllable glowing plant pet as a solution to help people alleviate anxiety in daily life. Therefore, we have considered various potential impacts on humans and the environment during the creation of this product. This year, we analyzed the risks that may arise throughout the project and outlined the following details.

We have seriously completed the Safety form, carefully reflected and reviewed the 34 questions mentioned in the official iGEM safety form. You are welcome to view the detailed safety form about our project submitted here！

In designing our safety protocols, since we will be using genetically modified glowing tobacco (FBP-T2 line/FBP-22 line) as the plant chassis for related research, we have implemented a series of measures to ensure the safety of the experiments and minimize risks. Below are the specific decisions we made in the design process:

During the course of the project, we received training on bioethics and safety, which allowed us to revisit and gain a deeper understanding of ethical principles. By studying and analyzing various ethical cases, we formulated the following guidelines.

1. Strict Selection of Non-pathogenic Chassis:

We chose to use non-pathogenic, commercially available Escherichia coli TOP10 and Agrobacterium GV3101 as microbial vectors to construct the chassis, and selected the commonly used model plant Nicotiana benthamiana as the plant chassis for functional validation. This ensures that no health risks are posed to humans, animals, or plants during the experimental process.

2. Selection of Components that Do Not Harm Humans, Animals, or Plants:

When selecting the vector backbone, we chose the pCAMBIA1300 backbone, which is free of any harmful effects. Components added to this backbone, such as plant promoters, terminators, the resistance marker gene Kan, and reporter genes like GFP/GUS, have all been widely used and are harmless to humans and the environment.

3. Substitute Hazardous Materials with Safer Alternatives:

In the validation experiments, we replaced traditional hazardous materials with safer alternatives commonly available in the market. For example, we used non-toxic or low-toxicity nucleic acid dyes instead of the cell-permeable ethidium bromide (EB) dye, and weak acid solutions to dissolve acetylacetone instead of using the toxic DMSO solvent.

According to the 'Regulations on the Safety Assessment and Management of Agricultural Genetically Modified Organisms,' the microorganisms TOP10 and GV3101, the genetically modified plant FBP-T2 line, and future transgenic products we study are classified as Biosafety Level I (non-dangerous), indicating no risk to the environment or humans. Therefore, in strict compliance with national and international laws, regulations, and standards, we successfully applied for and utilized the standard genetically modified plant research laboratory at the Shenzhen BGI Research Institute of Life Sciences. We have implemented the following safety measures:

1. Safe Genetic Modification Methods:

We use transient Agrobacterium-mediated transformation, which offers advantages such as no permanent genetic modification and low environmental risk. This method is simple, efficient, and does not require complex tissue culture and regeneration processes, thereby reducing risks of pathogen spread, contamination, and cross-contamination in the laboratory environment. During the transient transformation of tobacco leaves, plant materials do not require additional experimental treatments, so the injection operations are performed in a dedicated isolation chamber within the transgenic isolation climate room. After the experiment, thorough disinfection is conducted immediately to prevent any risk of spread and cross-contamination.

2. Controlled Enviroment Chamber Management:

The laboratory has separate controlled environment chambers for non-transgenic and transgenic materials, including areas for tissue culture, preparation, inoculation, washing and disinfection, sterilization, waste sterilization, and waste storage. Our transgenic tobacco FBP-T2 line materials are cultivated in a specific transgenic chamber, with detailed records maintained for material and personnel access to ensure that materials are not lost and genes do not escape. Non-transgenic materials such as Arabidopsis, periwinkle, ornamental roses, and cherry tomatoes are managed in standard climate chambers, completely isolated from transgenic materials. During tobacco leaf injection experiments, operations are conducted in a designated sample inoculation area, and thorough disinfection of the area is performed after the experiment.

3. Data Protection and Privacy:

Data Security: Ensure that all data and information related to genetically modified plants are protected in accordance with relevant laws and regulations to prevent data leakage and misuse.

Transparency: Disclose relevant information about genetically modified plant projects to ensure public right to information and participation.

4. Use of Enclosed Protective Equipment:

When transferring genetically modified plant materials for formaldehyde treatment, in addition to pre-registration and reporting procedures, we use standardized enclosed equipment for transport to ensure that the transgenic plants do not come into contact with the natural environment and are physically isolated for protection.

5. Biological Switch Mechanism:

Team iGEM14_Valencia_UpV 2014 submitted two biosafety devices for plants, BBa_K1554004 and BBa_K1554005, which specifically express barnase in anthers under the regulation of the TA29 tapetum-specific promoter to make the plants infertile. This will avoid the dispersion of the plant's genetic material in the environment. In the future, we aim to apply these two modules to the design of FBP enhanced plants for strict biosafety implementation.

6. Public Opinion Survey:

We have concerns about the use of genetically modified plants, as this topic may spark public controversy. As part of our human practice, we conducted a survey to gather public opinions on the use of genetic modification and incorporated these findings into our public outreach efforts. This approach has received positive feedback from the public during the outreach activities we participated in, aiming to minimize concerns and foster discussion on the topic.

We strictly adhere to local laws, regulations, rules, and guidelines, carefully managing the ways in which the project interacts with humans, as well as the collection and analysis of information. The project does not involve any human biomedical samples. Regarding the connection between plants and humans, we primarily focus on the following two aspects:

1. Informed Consent and Ethical Review:

Informaed Consnet: Our project research does not involve any human subjects. All team members have provided informed consent and are fully aware of the research objectives, procedures, potential risks, and benefits.

Ethical Review: All research projects have undergone rigorous review and approval by the BGI Ethics Committee to ensure compliance with ethical and legal requirements.

2. Regulatory Compliance:

Laws and Regulations: Ensure that all project experiments strictly comply with relevant national and international laws, regulations, and standards.

Safety Certification: Before the commercialization of genetically modified plants, obtain the necessary safety certifications and approvals. Currently, our materials are not involved in commercialization.

1. Environmental Impact Assessment:

Since the inherent hazard levels of the genetically modified plants or microorganisms we work with are very low, and the research is conducted in a relatively enclosed environment with proper handling before and after experiments, we ensure that the research process does not have a negative impact on the environment. All research activities comply with environmental protection regulations and standards.

2. Safe Collection of Environmental Samples:

When taking samples, we only collect plant tissues from artificially cultivated plants in the climate chamber for RNA or DNA extraction. During the process, we follow strict safety procedures to ensure that the sampling does not cause environmental contamination or damage. All sampling equipment and containers are disinfected and sterilized to avoid cross-contamination.

3. Sample Transport and Storage:

Except for plant materials that require short-distance transportation for formaldehyde treatment, no other samples involve outdoor transportation. Materials such as RNA, DNA, plasmids, E. coli, and Agrobacterium are involved in storage. These materials, especially plant materials, are strictly regulated and not taken out of the laboratory. They are transported in standardized enclosed equipment to a fume hood in the climate chamber inoculation area for processing. Other samples are stored in laboratory-grade refrigerators to prevent sample degradation or contamination, ensuring their integrity and safety.

We have successfully applied for and utilized the standard laboratory facilities at BGI Shenzhen for our project research. This facility provides comprehensive safety features and continuous supervision by mentors. The key aspects include:

Before entering the laboratory, we underwent safety training and foundational experimental knowledge with the staff of BGI Research Institute. This included simulations and practical skills for different laboratory activities, safety knowledge and case studies, and proper use of laboratory equipment. We were granted access only after passing the required assessment.

During this period, we strictly adhered to the Research Institute's internal 'Laboratory Safety Management Regulations' while conducting our projects. To ensure that team members work in a safer laboratory environment, we also complied with the iGEM competition's safety and security rules.

To avoid accidents or damage, we implemented appropriate safety measures during experiments, such as wearing gloves, masks, laboratory coats, and safety goggles when necessary, to protect against biological and non-biological hazards.

The following is a part of the safety training curriculum we covered:

At the same time, we adhere to a system of mutual supervision by at least two people. After completing daily experiments, we check the laboratory safety and standardize the behavior of laboratory team members. Experienced mentors also provide guidance and regulations on experimental operations and safety management to ensure that team members conduct experiments smoothly in a safer laboratory environment.

The biological laboratory we work in has a safety level of 1 (low risk) and is equipped with appropriate safety and emergency equipment, such as biosafety cabinets, emergency eyewash stations, incubators, sterilization devices, sinks, storage cabinets, electrical equipment, and fire-fighting equipment.

Reagents and chemicals are categorized and stored accordingly. Flammable and explosive chemicals (such as anhydrous ethanol, isopropanol, sodium hypochlorite, formaldehyde) are kept in dedicated storage cabinets with warnings and are locked with dual-person management. We first register and report when using these reagents, and the required amount is retrieved by the administrator. The reagents are then used in a designated fume hood for safe experimentation.

For specialized equipment (such as autoclaves for high temperature and pressure), we follow regulations by registering and reporting in advance and seek assistance from certified personnel to operate the equipment.

Our project involves handling hazardous chemicals such as formaldehyde, and the amount of formaldehyde used in experiments will be strictly controlled within safety limits. We adhere to safety protocols by preparing 2mM formaldehyde solutions, applying formaldehyde to samples, and sealing them in a chemical fume hood. Additionally, we wear personal protective equipment, including lab coats, safety goggles, masks, and gloves. Waste liquids, discarded materials, and sharp objects are collected and stored in designated hazardous waste areas and are subsequently disposed of by a professional hazardous waste management company.

Additionally, we place a special emphasis on the safety of microbiological experiments. This year, we use low-risk microorganisms, such as E. coli TOP10 and Agrobacterium GV3101, for basic biological experiments. For working with low-risk organisms, we conduct most experiments in a clean bench or biological safety cabinet to prevent aerosol generation. For products related to transgenic plants, we ensure that they are not cultivated in unintended locations and implement specific biological containment and isolation measures according to regulations to limit their spread. For waste materials related to microbiological experiments and transgenic plants, we first use chemical disinfectants for initial decontamination, followed by thorough sterilization using high-temperature and high-pressure methods in a designated waste sterilization area. The treated waste is then disposed of in specific bins and handled by approved disposal facilities.

The laboratory is equipped with emergency response equipment, including fire extinguishers, first aid supplies, and accident response materials. Detailed emergency response plans have been established to ensure swift and effective handling of safety incidents. During experiments, we have also developed emergency response procedures for laboratory safety incidents based on training, as outlined below:

1. Stop the experiment immediately: In the event of a safety incident, all experimental operations should be stopped immediately to ensure that the experimental equipment is in a safe state.

2. Assess the circumstances of the accident: Quickly assess the type and severity of the incident, including whether it involves chemical spills, fire, injuries, etc.

3. Notify relevant personnel: Immediately notify the laboratory supervisor and safety personnel to report the incident.

4. Evacuate personnel: Depending on the severity of the incident, evacuate all personnel from the laboratory immediately if necessary, ensuring they are kept away from the danger zone.

5. Use emergency equipment: use the emergency equipment provided in the laboraatory according to the type of incident. For example:

• Chemical spill: Use absorbent materials or a spill response kit to handle the situation.
• Fire: Use a fire extinguisher or fire blanket to extinguish the source of the fire.
• Luciferase (Luz): Catalyzes the oxidation of fungal luciferin, resulting in light emission.
• Injury: Use a first aid kit for initial treatment and call for emergency assistance as needed.

6. Ventilation and isolation: If there is a leak of toxic or irritating gases, the laboratory's negative pressure exhaust system should be activated immediately to ensure air circulation and isolate the contaminated area.

7. Cleaning and disinfection: After handling the incident, the affected area should be thoroughly cleaned and disinfected to ensure that no residual contaminants are left behind.

8. Documentation and reporting: Carefully record the time, location, cause, response process, and outcome of the incident, and submit an incident report to the relevant departments.

9. Incident analysis and improvement: Analyze the incident to identify its causes, develop improvement measures, and prevent similar incidents from occurring again.

We equip the laboratory with the necessary safety equipment and personal protective gear, and conduct regular safety training and emergency drills, either periodically or as organized by the research institute, to ensure that all team members are familiar with emergency procedures and the use of equipment.

Fortunately, we have focused on safety training and management, learning various key safety points and strictly adhering to the laboratory's rules and regulations to standardize experimental procedures. During this time, no safety hazards or incidents, large or small, have occurred. We consistently wear lab coats and gloves in the laboratory, disinfect our hands before leaving, and leave all supplies and products in the lab without taking them outside. We also avoid touching public areas with our gloved hands to prevent cross-contamination. This effectively reduces potential risks to public safety and health.

Through the integrated implementation of these design decisions and safety measures, we have minimized the potential risks present during the experimental process, ensuring the safety and reliability of the project, while also fully protecting the safety of both human and public environments.