Safety
Regarding the laboratory
Biosafety Law of China
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.
Training
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.
Basic Laboratory Rules
1.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.
2.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.
3.Designated individuals are responsible for ensuring that laboratory supplies are replenished in a timely manner to maintain the normal operation of the laboratory.
4.Smoking, eating, and sleeping are strictly prohibited within the laboratory.
5.The laboratory must be kept clean and organized, with waste promptly cleaned or disposed of.
Workplace Safety
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.
iGEM Rules and Policies
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.
Chassis and Parts Usage
Throughout our experimental work, the NEFU-China team did not use any organisms or parts classified as Risk Group 3 or 4. All laboratory work was conducted with Escherichia coli K-12 strains, specifically DH5α and BW25113, which are Risk Group 1 organisms widely used in research and considered safe for laboratory practice.The genes MNX1 PANAT and ABH60 were derived from Agaricus bisporus (button mushroom), a species that poses no risk to human health or the environment. In addition, the promoters and terminators we used do not present any hazards to humans or the environment.Therefore, all chassis organisms and genetic parts employed in this project fully comply with the 2025 iGEM White List requirements.
Laboratory Activities
The experimental activities of NEFU-China 2025 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.
Social Activities
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 interviewing professors and teachers or collecting survey questionnaires, our team ensured that all content complied with relevant legal and ethical standards, avoiding any potential social controversy.
About the Project
Microbial Safety
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
During our experimental work, we modified the E. coli BW25113 strain by knocking out the nhoA gene, thereby constructing the BW-ΔnhoA strain. The nhoA gene functions in a competing pathway that converts p-ABA into a by-product, p-acetylaminobenzoic acid, which diverts metabolic flux away from the AAP biosynthetic pathway. Therefore, knocking out nhoA suppresses this competing pathway and increases the yield of our target intermediate, 4-aminophenol (P-AP). To construct the BW-ΔnhoA strain, we employed a CRISPR-Cas9 knockout strategy. A donor plasmid (Donor-D500U500-nhoA) carrying the target sequence was introduced into BW25113 cells by electroporation, followed by screening to obtain successful BW-ΔnhoA colonies.
Our final target product, acetaminophen (AAP), is one of the most widely used antipyretic and analgesic drugs worldwide. Its major advantage lies in its mild effect on the gastrointestinal tract, making it suitable for patients who cannot take aspirin. Within the recommended dosage range, acetaminophen is considered safe for the vast majority of individuals.
The DNA manipulation techniques used in this study were based on homologous recombination and type II restriction endonuclease methods, both of which are well-established and safe approaches for DNA recombination. All laboratory operations were carried out under strict biosafety regulations to prevent any potential release of genetically modified microorganisms into the environment.
Reagent Safety
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.
