Laboratory Safety

Our team places laboratory safety and project safety as the foundation of our work. We strictly comply with national and school regulations, including the Biosecurity Law of the People’s Republic of China (2020) as well as Xi’an Jiaotong-Liverpool University’s Laboratory Safety Rules and Procedures Checklist Form. In addition, we provided all members with systematic laboratory safety training and assessments, enabling them to acquire the necessary technical skills, develop strong risk awareness, and handle hazardous chemicals and waste safely. Throughout the design and execution of the project, we continuously conduct risk assessments and use the Risk Assessment Form to identify and address potential hazards. Furthermore, we undergo regular laboratory safety inspections and consult biosafety, thereby ensuring the standardization and security of our experimental processes.

Training

Our project was conducted in the teaching laboratories of the School of Science at Xi’an Jiaotong-Liverpool University (XJTLU). Prior to the start of the project, all members completed an online laboratory safety training program, which covered an introduction to the laboratory, safety regulations, personal protective equipment (PPE), basic biosafety, and emergency response procedures. Upon completion of the training, all members were required to pass a biosafety examination before they were permitted to work in the laboratory. The examination covered topics including fundamental biosafety principles, response to chemical spills, fire safety, proper use of personal protective equipment (PPE), common laboratory instruments, and waste disposal.

After initial training, the wet-lab participants received two days of training on laboratory instrumentation. The training, conducted by Laboratory Manager Dr. Zhongkai Huang, covered a range of essential equipment, including but not limited to high-speed centrifuges, autoclaves, biosafety cabinets, cold rooms, cell culture incubators, and -20°C and -80°C freezers. In parallel, all students signed the Risk Assessment Form, ensuring their awareness and preparedness for potential risks. The laboratory also performs monthly safety inspections to minimize hidden risks. Throughout the entire experimental period, students are subject to continuous supervision and random safety checks by the laboratory manager, thereby ensuring both the standardization and the security of all experimental activities.

Before the start of the experiments, all wet-lab members received systematic training in early June for essential laboratory skills. The training covered fundamental molecular biology techniques, including gel electrophoresis, PCR, preparation of competent cells, electroporation, and chemical transformation. Under the guidance of the laboratory manager, members also became familiar with the storage locations and hazard classifications of laboratory chemicals. Regarding waste management, all laboratory waste is collected and sterilized by autoclaving before disposal, while liquid media containing bacteria must be disinfected prior to release at designated sites. To ensure safe operation, the use of the autoclave is carefully documented, including the time of use, materials sterilized, and the operator’s name, with records verified by the laboratory manager. Team members involved in cancer cell culture received additional training covering cell thawing, cryopreservation, passaging, and flow cytometry.

Safety Rules

During the project, the laboratory follows strict safety regulations. To ensure personal safety, at least two team members must be present for any nighttime work in the lab. Eating，drinking and storing any other non-laboratory items are strictly forbidden. Team members are required to wear lab coats and gloves, and safety goggles when necessary. In addition, for safety reasons, members with long hair must keep it securely tied back while working in the laboratory.

Biosafety Discussions

During the design and development of our project, we held discussions on biosafety with several professors from the School of Science and the academy of Pharmacy at Xi’an Jiaotong-Liverpool University who specialize in cancer or microbiology. The primary goal of these discussions was to ensure the feasibility and compliance of our project, while identifying and solving potential safety risks at the early stage. In this process, the professors raised several important concerns regarding the chassis bacteria we selected. Their questions centered on three aspects: first, whether the strains could be classified as probiotics and demonstrated compatibility with humans and the environment; second, whether they carried any potential pathogenic risks that might pose threats to laboratory personnel or the external environment; and third, whether such strains could gain acceptance from the general public, thereby enhancing the applicability and social recognition of our project. In addition, discussions with Professor Youming Zhang from Shandong University introduced us to the special strain E. coli Nissle 1917. These experiences provided us with valuable guidance. By consulting with experts, we got a deeper understanding of the biosafety of our chassis strains and realized the importance of public acceptance. As a result, we are placing greater emphasis on strain selection and risk assessment in our ongoing work to ensure full compliance with biosafety standards.

Safe Research Design

Ultimately, we adjusted our choice of chassis strains. Instead of using Staphylococcus aureus, which has strong infectivity but high pathogenicity, we selected three safer strains: Staphylococcus xylosus ATCC 29971, Staphylococcus epidermidis ATCC 14990, and Escherichia coli Nissle 1917. Among these, E. coli Nissle 1917 is widely recognized as a probiotic and is clinically used for treating gastrointestinal diseases such as IBD and UC. S. epidermidis ATCC 14990, meanwhile, is classified as a low-risk, BSL-1 strain. Given these, we constructed a hypoxia-inducible promoter and a “suicide switch” designed for the hypoxic microenvironment of tumors. The hypoxia-inducible promoter (Pnart) specifically triggers expression of toxin protein under hypoxic conditions, while remaining inactive in aerobic environments such as intercellular spaces or normal tissues. Furthermore, by linking Pnart to a resistance gene (e.g., ermC), the system is activated within tumor cells but remains dormant elsewhere. This strategy enables precise targeting and killing of tumor cells while minimizing damage to surrounding healthy tissues. Finally, toxin protein selection was carefully evaluated to reduce potential harm to normal cells. We introduced Apoptin, a protein derived from the chicken anemia virus, as our specific killing effector. Notably, apoptin exhibits unique selectivity for cancer cells, while in normal cells it remains sequestered in the cytoplasm and largely inactive.