Safety

Safe Project Design

Great importance is attached to safety in our project design.

Firstly, we use E. coli as our chassis organisms, which poses no harm to individuals or environment. E. coli DH5α, DH10β, BL21(DE3), JM109(DE3), MG1655, and BL21-AI are utilized when we implement the experiments of molecular cloning, expression and verification.

This year, our project aims to efficiently degrade chitosan into chitooligosaccharides (COS) with enhanced antibacterial activity, and to develop a natural seafood preservative. To achieve the efficient degradation of chitosan, we constructed a chitosanase mutant library through random mutagenesis and expressed the mutant enzymes in E. coli JM109(DE3). To bypass the expensive enzyme purification step, we utilized signal peptides to secrete chitosanase into the supernatant. This supernatant was then used to degrade chitosan for COS production. After supernatant collection, the remaining cell pellet was immediately autoclaved to prevent any release of the engineered strain. In the preservative application assays, we used only the COS-containing solution, with no engineered bacteria involved. To prevent the spread of engineered strains in the environment, we designed a dual-safeguard kill switch system composed of a cuminic acid-regulated ccdB/ccdA toxin-antitoxin module and an IPTG-inducible tyrS overexpression module. Once the engineered bacteria have completed their function, the addition of cuminic acid and IPTG activates the kill switch, leading to cell death. The two systems operate independently and provide mutual backup, ensuring rapid cell death and minimizing the risk of strain escape.

All the proteins are totally harmless to human body. All the plasmids are harmless for human beings and none of our parts would raise any safety issue according to the current expertise.

In addition, we performed microbial colony count analysis to evaluate the preservative efficacy. This process might involve the isolation and cultivation of potentially pathogenic microorganisms. To ensure experimental safety, all procedures involving biosafety level 2 (BSL-2) microorganisms were conducted in a BSL-2 laboratory. Approved by the Safety and Security Committee (Safety Form), we strictly followed all approved BSL-2 procedures to minimize the risk to both personnel and the environment.

Safe Lab Work

The laboratory is equipped with the location of emergency exits, emergency ventilation devices, fire-fighting equipment, first aid kits, and so on. Every year after recruiting new members, we spend several weeks training them in basic laboratory operations and handling safety accidents. When entering the laboratory, we must wear laboratory clothes, gloves, and goggles. All bacterial culture mediums have undergone sterilization treatment. We strictly follow the BSL-1 regulations to dispose of all biological materials and metabolic waste. The laboratory is also equipped with specialized operators for handling hazardous materials and biological products.

Figure 1 First aid kits of our lab.

At the same time, we invited laboratory safety engineer, Lina Wu, to conduct safety education for all team members when they officially got into the experimental phase.

Figure 2 Members learnt laboratory safety intentions.

During the microbial cultivation and analysis phase of the preservation experiment, BSL-2 microorganisms may be isolated. Therefore, it is essential to strictly adhere to the corresponding biosafety regulations. This experiment will be conducted in a BSL-2 laboratory within the shared research facilities. The laboratory fully complies with national BSL-2 standards and is equipped with buffer rooms, HEPA filtration, directional negative pressure ventilation, and multiple Class II biosafety cabinets to prevent aerosol leakage and cross-contamination. Any contaminated waste or used materials generated will be immediately autoclaved on-site prior to further handling or disposal, in strict accordance with established protocols, ensuring complete inactivation of all biological materials before leaving the laboratory.

Furthermore, all team members have completed comprehensive laboratory safety training, covering biosafety regulations, BSL-2 laboratory protocols, proper use of personal protective equipment (PPE), biosafety cabinet operation, autoclave procedures, emergency response, and waste disposal. All participants have passed the required assessment.

Figure 3 (A) The anteroom of the shared BSL-2 laboratory.
(B) The biosafety cabinet in the shared BSL-2 laboratory.