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Safety

Catalog

Introduction

Safety is the core principle of our experimental project and the foundation for its success. To ensure smooth experimental procedures and the health and safety of our team members, we strictly adhere to safety protocols at every stage of the project. We recognize that in the field of synthetic biology, safety is not only a project requirement but also a responsibility toward society and the environment. Therefore, from the selection of chassis organisms and project design to experimental operations, rigorous safety strategies are applied at every step.

Before the project started, we conducted a comprehensive assessment of all potential biological and chemical risks to ensure that all materials and procedures met safety standards. Our team not only adheres to the highest safety standards during experiments but also places special emphasis on raising safety awareness and enhancing the skills of its members. All team members have undergone thorough safety training and passed the laboratory access permit exam, ensuring their competence in safe laboratory operations.

Through these measures, we have ensured the safety of our experiments and laid a solid foundation for the project's success. Our goal is to conduct high-quality scientific research while setting a model of excellent safety management for future synthetic biology projects.

Microbial Safety

Our chassis organism, Aureobasidium melanogenum BZ-11, is classified as BSL1 and has been approved through the iGEM Check-In Form. According to general principles of microbial and biomedical laboratory biosafety, BSL1 applies to microorganisms known to pose no pathogenic risks to healthy adults. Aureobasidium spp. has been widely used in synthetic biology, and extensive research has been conducted on it over the years [1]. We note that many other chemical production processes based on Aureobasidium melanogenum have been proven safe [2][3].

Figure 1 Aureobasidium melanogenum BZ-11 has been approved through the iGEM Check-In Form

In addition, we use Escherichia coli DH5α as a cloning vector in our experiments. E. coli DH5α is a commonly used competent strain for cloning in biology and is on the whitelist of approved organisms. Therefore, we strictly follow the safety protocols for BSL1 laboratories in our operations and management.

Project Design Safety

We have also taken numerous precautions in our project design to ensure its safety:

1. In our project, we knocked out the PKS gene. The PKS gene is critical for melanin synthesis and is also important for the synthesis of other secondary metabolites, significantly influencing the fungus's pathogenicity and survival in the environment [4]. After successfully knocking out the PKS gene, we not only halted melanin synthesis, producing a purer product, but also greatly reduced the strain's ability to survive in the wild, further preventing accidental release.

2. During gene knockout, we did not retain the antibiotic resistance gene. The knockout vector used the plasmid pFL4A-NAT-loxp carrying the nourseothricin resistance gene (NAT gene). After successfully screening for strains with the desired gene knockdown, we performed counterselection by using a plasmid carrying the Cre recombinase gene to remove the NAT gene. This gene is automatically lost during yeast cell division, and we subsequently conduct replica plating to screen for strains that have undergone successful gene knockdown and do not contain antibiotic resistance [5][6].

Figure 2 Conceptual diagram of the gene knockout method

Laboratory Safety

1. Relevant Laws and Regulations

All project personnel must strictly comply with the 2021 Biosafety Law of the People's Republic of China and the Laboratory Safety Guidelines for Higher Education Institutions issued by the Ministry of Education of the People's Republic of China. The Biosafety Law of China outlines requirements for risk assessment, management, and emergency measures in biological experiments, covering the handling, transportation, and storage of biological materials. The Laboratory Safety Guidelines provide detailed requirements for laboratory environment setup, equipment usage, and experimental operation protocols, ensuring that laboratory activities meet safety standards and prevent accidents. These laws and regulations aim to ensure the normative operation of laboratories and protect researchers and the environment from potential hazards [7][8].

2. Laboratory Safety Guidelines

Our team strictly follows BSL1 (Biosafety Level 1) laboratory safety protocols to ensure the safety and hygiene of the laboratory environment. Specific guidelines include:

a) Laboratory Environment:

Design and Layout: Laboratories should have proper ventilation, typically including natural or mechanical ventilation systems. The lab should be an open workspace without specialized ventilation systems, suitable for handling low-risk biological materials.
Facilities and Equipment: Workbenches should be durable and easy to clean. Laboratories should be equipped with basic experimental equipment, such as incubators and centrifuges, which must be regularly maintained and kept in good condition.
Cleaning: Surfaces should be regularly cleaned and disinfected, especially after experiments. Periodic deep cleaning of floors, walls, and counters is essential to prevent the buildup of contaminants.

b) Personal Protective Equipment (PPE):

Lab Coats: Personnel should wear appropriate lab coats to prevent direct contact between experimental materials and skin. Lab coats should be kept clean and changed regularly.
Gloves and Goggles: Disposable gloves and goggles should be worn when handling materials that may generate aerosols or splashes. Although BSL1 risks are low, these measures further reduce the risk of accidental exposure.
Hairnets and Covered Shoes: While not strictly required for BSL1 labs, it is recommended that personnel wear hairnets and covered shoes to reduce potential contamination from hair and shoe soles.

c) Laboratory Operation Protocols:

Work Area: Keep materials, tools, and equipment neatly arranged on the bench to maintain a clean and organized workspace.
Disinfection and Cleaning: Workbenches should be disinfected after each experiment. Hands should be cleaned and disinfected before and after experiments.
Waste Disposal: Biological waste (e.g., culture media, disposable lab supplies) should be disposed of according to prescribed procedures. Liquid waste can be treated with autoclaving or chemical disinfection, while solid waste should be handled according to laboratory waste management guidelines.

d) Entry and Exit Protocols

Personnel must complete personal cleaning and disinfection before entering the lab. No food, drinks, or cosmetics are allowed to maintain a clean environment. Personnel should be in good health, free of infectious symptoms. Those with contagious diseases, such as colds or coughs, should avoid entering the lab to prevent the spread of pathogens.

e) Laboratory Safety Management

Training: All personnel must undergo appropriate safety training, covering laboratory protocols, emergency procedures, and accident reporting processes.
Safety Records: The lab must maintain detailed safety records, including experimental logs, equipment maintenance records, and accident reports.

f) Emergency Procedures

Emergency Facilities: The lab should be equipped with basic emergency facilities, such as eyewash stations, emergency showers, and first-aid kits, to address potential incidents during experiments.
Accident Reporting: There should be an accident reporting and handling procedure to ensure that all incidents (e.g., chemical spills, biological material leaks) are promptly addressed and recorded. All accidents should be thoroughly documented and analyzed to prevent recurrence.

g) Records and Documentation

Operational Logs: The lab should maintain logs documenting experimental activities, equipment maintenance, and safety inspections. Logs should include details of all experiments to facilitate tracking and review.
Safety Documentation: The lab should keep all relevant safety documents, such as laboratory protocols, safety data sheets, and training records, for easy management and reference.

Figure 3,4 Our team member in the lab

3. Chemical Safety

Chemical Procurement and Storage: Chemicals must be purchased from suppliers that meet safety standards, and labels and instructions must be complete. Chemicals should be stored under appropriate conditions, such as temperature and humidity, as specified in the instructions to prevent degradation or spoilage.
Use of Chemicals: Chemicals must be handled according to operating procedures, with appropriate PPE worn, and used in well-ventilated environments. Hazardous chemicals should be used in dedicated safety cabinets with additional protective measures.
Chemical Waste Disposal: Chemical waste must be treated according to laboratory waste management regulations, avoiding mixing with regular waste. Designated waste containers should be used for collection, and disposal should follow specified procedures.

Figure 3 Ocean University of China Laboratory Safety Guidelines

Member Safety Training

A year before the project officially began, our team underwent a two-week laboratory safety access training and passed the laboratory access permit exam conducted by the university. Before starting the experiments, we also conducted focused training on experimental skills and related precautions.

1. Laboratory Safety Access Training

a) Basic Laboratory Safety Knowledge

Chemical Safety: Learn to identify and handle commonly used chemicals in the lab, understand the use of Safety Data Sheets (SDS), and comprehend chemical classifications, labeling, and associated risks.
Biosafety: Understand the differences between biosafety levels (BSL 1-4), proper handling of biological samples, and protocols for using biosafety cabinets.
Personal Protective Equipment (PPE): Learn the proper use and importance of common laboratory PPE, such as lab coats, gloves, and goggles.
Emergency Response: Procedures for handling chemical spills, fires, electric shocks, and lab injuries, and the location and usage of emergency equipment like eyewash stations, fire extinguishers, and first-aid kits.

c) Laboratory Ethics and Compliance

Research Ethics: Learn the ethical guidelines for animal experimentation, human sample usage, and the principle of informed consent.
Compliance Requirements: Understand and adhere to relevant school and national laws and regulations, as well as the approval procedures and ethical review process for research projects.

d) Specific Laboratory Skills

Aseptic Techniques: Master the basic steps of aseptic technique and skills to prevent contamination during experiments.
Experimental Design and Execution: How to design experiments properly, control variables, and the importance of reproducibility and accuracy.
Data Analysis and Interpretation: Basic statistical analysis methods, how to interpret experimental results correctly, and how to write a report.

e) Case Studies and Applications

Accident Case Analysis: Learn from historical laboratory accident cases, analyze their causes, and how to avoid similar incidents.
Practical Scenario Drills: Propose solutions for hypothetical scenarios, such as how to handle a chemical spill or protect experimental data during a power outage.

2. Laboratory Access Permit Exam

Practical Operation Exam: Assess trainees' proficiency in using lab instruments, wearing PPE, and handling waste through practical demonstrations.
Online Assessment: Evaluate trainees' knowledge of lab safety, regulations, and operational skills through multiple-choice, short-answer, or case analysis questions.
Simulation Platform Assessment: Using the school's official online platform, simulate common laboratory emergencies to test how students would respond to sudden incidents.

References

[1] Wang, P., Jia, S.-L., Liu, G.-L., Chi, Z., & Chi, Z.-M. (2022). Aureobasidium spp. and their applications in biotechnology. Process Biochemistry, 116, 72–83. https://doi.org/10.1016/j.procbio.2022.03.006
[2] Chen, G., Zhu, Y., Zhang, G., Liu, H., Wei, Y., Wang, P., Wang, F., Xian, M., Xiang, H., & Zhang, H. (2019). Optimization and characterization of pullulan production by a newly isolated high-yielding strainAureobasidium melanogenum. Preparative Biochemistry & Biotechnology, 49(6), 557–566. https://doi.org/10.1080/10826068.2019.1591988
[3] Zhou, R., Ma, L., Qin, X., Zhu, H., Chen, G., Liang, Z., & Zeng, W. (2023). Efficient Production of Melanin by Aureobasidium Melanogenum Using a Simplified Medium and pH-Controlled Fermentation Strategy with the Cell Morphology Analysis. Applied Biochemistry and Biotechnology, 196(2), 1122–1141. https://doi.org/10.1007/s12010-023-04594-8
[4] Eisenman, H. C., Greer, E. M., & McGrail, C. W. (2020). The role of melanins in melanotic fungi for pathogenesis and environmental survival. Applied Microbiology and Biotechnology, 104(10), 4247–4257. https://doi.org/10.1007/s00253-020-10532-z
[5] Chen, T.-J., Liu, G.-L., Wei, X., Wang, K., Hu, Z., Chi, Z., & Chi, Z.-M. (2020). A multidomain α-glucan synthetase 2 (AmAgs2) is the key enzyme for pullulan biosynthesis in Aureobasidium melanogenum P16. International Journal of Biological Macromolecules, 150, 1037–1045. https://doi.org/10.1016/j.ijbiomac.2019.10.108
[6] Goldstein, A. L., & McCusker, J. H. (1999). Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast, 15(14), 1541–1553. https://doi.org/10.1002/(sici)1097-0061(199910)15:14%3C1541::aid-yea476%3E3.3.co;2-b
[7] Biosecurity Law of the People’s Republic of China. (2020). Cdurl.cn. http://en.npc.gov.cn.cdurl.cn/2020-10/17/c_703568.htm
[8] Notice of China’s Ministry of Education on the issuance of the Measures for the Administration of Laboratory Safety Classification in Universities and Colleges (Trial Implementation)_ Department Documents of The State Council _ China Government Net. (2024). Www.gov.cn. https://www.gov.cn/zhengce/zhengceku/202404/content_6946788.htm