All project personnel strictly comply with the Biosafety Law of the People's Republic of China (2024 Amendment), which was implemented in 2021 and revised for the first time on April 26, 2024.
Before formally commencing laboratory experiments, all members of the project underwent a two-month theoretical training. They also participated in intensive training on experimental skills and associated safety precautions.
- Before beginning work, it is essential to clearly identify the locations and operating procedures for fire extinguishers, electrical switches, and other facilities. Familiarity with emergency measures and procedures is also required.
- Laboratory coats must be worn upon entering the laboratory, and flat-soled shoes should be avoided. When conducting experiments that may involve risks, appropriate personal protective equipment, such as safety goggles, face shields, and protective gloves, must be worn as needed. Laboratory coats should be removed before leaving the laboratory.
- Designated individuals are responsible for ensuring that laboratory supplies are replenished in a timely manner to maintain the normal operation of the laboratory.
- Smoking, eating, and sleeping are strictly prohibited within the laboratory.
- The laboratory must be kept clean and organized, with waste promptly cleaned or disposed of.
To prevent contamination, exposure, or accidental release of genetically modified organisms, laboratory facilities must meet the specific requirements of the experiments. Below, you will find photographs of the laboratory. The work is conducted in the following laboratory:
Northeast Forestry University, Room 235, Yifu Building
Regular cleaning of the laboratory bench is done by spraying with a 75% alcohol solution. During sterile operations, personnel must ensure that no flammable materials are brought near the open flame of the alcohol burner. Ultraviolet sterilization will be performed on the clean workbench before and after conducting microbiological experiments.
During agarose gel electrophoresis and SDS-PAGE experiments, reagents, instruments, and other contaminated items are separated from uncontaminated items, creating a designated contamination zone. When working within this contamination zone, personnel adhere to stricter protective measures to ensure their safety.
The majority of laboratory workspace operates at Biosafety Level 2 (BSL-2), which is moderately sealed. All tasks are performed within open workbenches, chemical fume hoods, or biological safety cabinets.
The iGEM rules and policies provide effective guidelines for risk management. By responsibly implementing these regulations, researchers ensure the compliance of their projects. The following outlines specific measures taken to adhere to these rules and policies.
Throughout NEFU-China's experimental process, no organisms or parts from Risk Group 3 or Risk Group 4 were used. The experiments primarily utilized strains of Escherichia coli K-12, namely DH5α and BW25113, which belong to Risk Group 1 and are commonly used in research. The ndm gene was sourced from Pseudomonas putida CBB5, a strain harmless to human health and the environment. The promoters and terminators used also pose no threat to human health or the environment. Therefore, all chassis and parts are in compliance with the iGEM 2024 white pape.
The experimental activities of NEFU-China 2024 did not involve any animals, plants, or human samples, and no experiments related to human, animal, or plant bodies were conducted. No biological samples were isolated from the environment, nor were any organisms obtained from sources outside of trusted commercial or institutional suppliers, thus avoiding gene drives. Additionally, no genetically modified organisms or their products, or new resistance factors, were deployed outside the laboratory. Upon completion of experiments, waste bacteria were disinfected and toxic reagents were appropriately disposed of in accordance with regulations. Therefore, no activities prohibited by the competition were carried out.
The iGEM team regularly engages with the community to gain a deeper understanding of broad social issues. All social activities strictly comply with local laws and regulations. For example, when collecting public opinions and psychological data, the team ensured that the content adhered to relevant legal and ethical standards, avoiding any potential social controversy.
In the current project, the chosen chassis strains are Escherichia coli K-12, specifically the competent strains DH5α and BW25113. According to the ATCC, both strains can be used in a BSL1 laboratory. The general principles of biosafety for microbiology and biomedical laboratories specify that BSL1 is appropriate for microorganisms known to pose no pathogenic risk to healthy individuals. Escherichia coli K-12 is also listed in iGEM’s "White List" and is classified under Risk Group 1.
DH5α
BW25113
K-12
In constructing the 7-MX whole-cell biosensor, the BW25113 strain was modified by knocking out the guaB gene to create the BW-ΔguaB strain. The guaB gene is responsible for converting IMP to XMP, an important substrate for DNA and RNA synthesis in Escherichia coli. Consequently, the knockout of the guaB gene impairs DNA and RNA synthesis, leading to cell death. To construct the BW-ΔguaB strain, the RED homologous recombination method was used, employing the pKD46 plasmid for the knockout. A pKD46 plasmid containing the targeting fragment was prepared, transformed into BW25113 via electroporation, and plated. The successful BW-ΔguaB strains were then selected from the plates.
The synthetic product 7-MX is a natural metabolite of caffeine known for its potential to alleviate myopia and its long-term safety.
The DNA manipulation techniques used in the experiments are based on homologous recombination and type II restriction endonuclease technology, both of which are safe and reliable DNA recombination methods. The laboratory enforces strict adherence to safety guidelines to prevent any contamination of the environment by genetically modified microorganisms.
The laboratory provides various safety equipment, including face washers, eye wash stations, first aid kits, medical supplies, fire extinguishers, and sand. All purchased experimental reagents adhere to strict safety standards. For example, the team opted for Goldview as the nucleic acid dye instead of using highly carcinogenic ethidium bromide.
