This project aims to develop an efficient cancer treatment strategy by utilizing Escherichia coli BL21(DE3) as the host strain to construct an engineered system regulated by the ALPaGA lactate-responsive promoter, enabling the specific expression of canine kynureninase (KYNase) in the tumor microenvironment. This expression facilitates the degradation of KYN molecules, thereby alleviating immune suppression and enhancing antitumor effects. All experiments are conducted in a standard microbiology laboratory, strictly adhering to safety protocols to ensure personnel safety and environmental protection.

• Standard Microbiology Laboratory: The experimental facility is equipped with biosafety cabinets and chemical fume hoods for handling bacterial cultures and sensitive materials, ensuring a safe operational environment.

• Personal Protective Equipment (PPE): All members must pass a safety examination to enter the laboratory and wear standard protective gear such as gloves, lab coats, and goggles throughout the experimental process.

• Waste Management: We strictly adhere to national laboratory waste classification and disposal regulations, separating biological and chemical waste for processing to minimize impact on personnel and the environment.

(1) Risk Management Measures

• Environmental Isolation: All experiments in this project are conducted in closed laboratories, with no release of engineered bacteria into the environment, ensuring that the research does not interfere with the external ecosystem.

• Standardized Operating Procedures: Team members strictly follow operating procedures during experiments to control contamination risks and reduce the probability of experimental accidents.

• Classified Waste Disposal: Experimental waste liquids, bacterial cultures, and equipment are recycled and inactivated according to type to ensure they pose no harm to human health or the environment.

(2) Biological Organism Management and Spread Risk

Engineered bacteria pose a minimal risk of accidental release. To address this, we implement multiple management measures, including pre-experiment safety training for all personnel, documentation of operational procedures, and closed management of the experimental area, to minimize the possibility of leakage.

(3) Experimental Standards and Training Mechanisms

• All team members have completed laboratory safety training and are proficient in microbiological experimental procedures, disinfection and sterilization processes, and emergency response protocols.

• Experiments are subject to standardized supervision throughout the process by the supervising instructor and the institution's biosafety officer to ensure compliance and safety.

Team members are being educated on lab safety

(4) Emergency Response Mechanism

The laboratory is equipped with first-aid kits and eye wash stations to provide timely treatment in case of accidents, ensuring the safety and health of personnel.

first aid supplies

This project is supported by the Laboratory Construction and Management Office of Nanjing University of Technology:

• Professor Liu Guannan, the principal investigator, provides comprehensive guidance on experimental procedures and safety protocols, with extensive experience in genetic engineering and laboratory management;

• Zhang Yabin, the university's biosafety officer, offers systematic experimental safety support, having organized multiple university-level safety trainings, emergency drills, and equipment inspections, with rich practical experience and numerous safety management awards.

Mr. Yabin Zhang, the institutional biosafety officer from Nanjing Tech University

We consistently adhere to the principle of “safety first,” fostering a strict safety culture within the team to ensure controllable experiments, transparent processes, personnel health, and an uncontaminated environment.

In Integrated Human Practices, we focus on “enhancing public awareness + risk education” as core objectives, conducting various outreach and feedback activities centered on synthetic biology safety:

• Public questionnaire survey and risk perception sampling

The team conducted risk perception and treatment acceptance surveys targeting different age groups, receiving over 100 valid responses. The findings revealed that most respondents harbored “uncertainty” concerns about living therapy, emphasizing the need for a balance between efficacy and side effects.

• In-depth interviews with families affected by disease

Through a series of interviews with two families of esophageal cancer patients, we collected feedback indicating a strong demand for new therapies such as “side effect reduction” and “home treatment,” which was used to optimize the design of the project's subsequent control system.

(1) Policy interviews: Expert symposium with the National Medical Products Administration

During the research process, we conducted interviews and consultations with experts from the National Medical Products Administration and the Jiangsu Provincial Drug Administration to gain a deeper understanding of the regulatory approval process for living cell therapy in China, as well as considerations related to biosafety and ethical boundaries.

Experts noted: “The regulation of synthetic biological therapeutic drugs is becoming increasingly stringent, with particular focus on the control, elimination, and traceability of engineered bacteria.” This feedback directly prompted us to prioritize the “biosafety module” in our design.

To prevent the risk of biological contamination caused by residual or leaked engineered bacteria after treatment, we designed and constructed a few biosafety modules:

• ALPaGA-inducible promoter module + PhiX174E lysis module: When bacterial concentration reaches a set threshold, the lysis mechanism is spontaneously activated to release therapeutic factors and eliminate itself;

• pDawn Blue Light-Induced MazF System: Upon exposure to blue light, MazF expression is activated to inhibit the continued growth of residual strains, ensuring safety in the in vitro environment.

This dual-pathway mechanism combines “density sensing + light signal control” as dual switches, ensuring therapeutic efficacy while achieving automatic clearance of engineered bacteria after treatment, thereby safeguarding both human and environmental safety.

We solemnly commit that the project will strictly adhere to national and institutional laboratory biosafety regulations, continuously improving experimental procedures and risk control systems. In the future, we will:

• Continuously optimize the suicide module and control mechanisms to enhance safety performance;

• Explore the use of human-derived proteins to replace exogenous proteins, reducing the risk of immune reactions;

• Organize more public science popularization activities to promote societal understanding and consensus on synthetic biological safety;

• Adhere to advancing project research within a framework of safety, standardization, and science, ensuring that technological development aligns with social responsibility.