In our 2025 project, we will genetically engineer mammalian cells (HEK293T cells) to release proteins of interest upon sensing chemical or light signals. This will be achieved by transfecting them with plasmids expressing our engineered protein, which anchors target protein-containing vesicles to the cytoskeleton. Upon receiving a specific signal, this engineered protein is cleaved or dissociated.

Throughout the project's design and implementation, we have consistently prioritized safety and addressed the following key considerations:

1. Standard molecular cloning utilized E.coli strains (DH5α and Stbl3). According to the literature, the strains used in this study are non-pathogenic to immunocompetent adults, thereby minimizing potential risks to laboratory personnel and the environment.
2. HEK293T cells were used to validate the functionality and efficiency of newly constructed plasmids. The HEK293T cell line is derived from HEK293 cells and incorporates the E1A gene from adenovirus. Although adenovirus can cause human illness, HEK293T cells are recognized as safe for laboratory use, as they cannot survive in uncontrolled environments outside the lab.
3. All genes cloned into the pcDNA3.1 vector were in a non-fusion format, effectively preventing the horizontal transfer of genetic material. We used the CMV promoter, ensuring our bacterial system does not produce significant amounts of the gene products without induction.
4. We have ensured that all biological materials used are safe for humans and will not cause environmental bio-contamination.

Although the potential hazards posed by E.coli DH5α and Stbl3 strains to laboratory personnel and the environment are minimal, it is necessary to acknowledge the risk associated with the potential spread of antibiotic resistance genes. As these cells typically harbor plasmids containing antibiotic resistance selectable markers, improper disposal of bacterial waste could lead to the dissemination of these genes. Therefore, we have implemented corresponding containment measures to prevent such contamination.

HEK293T cells are unlikely to survive outside the laboratory. However, the accidental introduction of these cells into the bloodstream could potentially lead to adverse health effects. Therefore, laboratory personnel must exercise extreme caution when handling them.

Furthermore, the cells used in this study retain the potential for division. The ethical review for such cells requires greater stringency. To manage potential risks, we have imposed strict limitations on the proliferation of engineered cells. To minimize any residual risk, further experimental investigation is necessary to enhance the overall safety and efficacy of our work.

We also use several chemicals in our experiments that may pose health hazards if mishandled. These include Diethanolamine (DEA), Dimethyl Sulfoxide (DMSO), Dexamethasone, and Polyethylenimine (PEI), which are associated with irritant, toxic, or sensitizing effects. Appropriate PPE, ventilation, and chemical waste disposal protocols are strictly followed to mitigate these risks.

1. Our team's approach to biosafety and risk management is grounded in national regulations, institutional guidance, expert oversight, and established laboratory practices. We adhere to the Biosecurity Law of the People's Republic of China, which governs biotechnology research, laboratory containment, and risk classification. Institutionally, we comply with our university's detailed biosafety protocols for handling genetically modified organisms, chemical safety, waste disposal, and personnel protection. These rules are enforced under the oversight of our Laboratory Biosafety Committee and institutional biosafety officer, whom we consult regularly.
2. All team members have completed mandatory biosafety training, covering lab conduct, PPE use, emergency response, chemical handling, and the use of containment equipment.Personnel must pass an assessment to obtain permission for independent work in the BSL-1 laboratory. A specific additional training program is required for personnel needing to work in the BSL-2 laboratory, which is used for mammalian cell culture, where the highest safety standards are followed.

3. Additional project-specific briefings addressed HEK293T cell engineering and antibiotic resistance gene containment. We work in BSL-1 and BSL-2 compliant laboratories, equipped with certified biosafety cabinets (Class II), chemical fume hoods, autoclaves, spill kits, and access controls. These facilities are routinely maintained and inspected.

4. To prevent environmental release and exposure, we implement strict waste inactivation procedures. Bacterial and mammalian cell cultures are decontaminated via autoclaving or chemical treatment (e.g., 10% bleach) before disposal. Chemical waste is collected separately and disposed of through licensed hazardous waste channels. All genetically modified organisms are securely stored, and inventory is tightly controlled. We also employ administrative controls like incident reporting systems and restricted access to hazardous agents.
5. Personnel must pass an assessment to obtain permission for independent work in the BSL-1 laboratory. A specific additional training program is required for personnel needing to work in the BSL-2 laboratory, which is used for mammalian cell culture, where the highest safety standards are followed.
6. Before using any new equipment, we carefully and repeatedly review the operating procedures to ensure proficiency. Initial independent operation must be performed under the direct supervision of an instructor.
7. We maintain an accident reporting system to document and learn from any lab incidents. Throughout the project, we have consulted with our institutional biosafety officer to evaluate potential risks, improve countermeasures, and ensure compliance. In response to identified risks, we have modified experimental designs to use an FDA-approved drug as the chemical inducer and decided not to pursue animal experiments.
8. Our work is supported by our institution's Laboratory Biosafety Committee, which oversees risk assessment and ensures regulatory compliance. If we identify a new hazard, we would promptly consult both the Biosafety Officers and our Principal Investigators to evaluate the situation and implement appropriate safety measures.