Safety and Security

1. Overview

From project conception to experimental execution, safety has remained the cornerstone of our research philosophy. During the design phase, every genetic element and experimental condition was evaluated through the lens of biosafety to ensure that no step poses potential risks to researchers or the environment. In the laboratory, all operations are performed under certified conditions, following standardized protocols and clearly defined emergency procedures. Team members receive comprehensive training in laboratory safety and research ethics before participating in experiments, fostering a culture of caution and accountability. Furthermore, all biological materials are securely contained and thoroughly sterilized after use to prevent unintended release. Through these integrated safety measures, our team ensures that scientific innovation proceeds hand in hand with responsibility, ethics, and environmental protection.

2. Part

After extensive literature research, we selected the toxin–antitoxin module based on the Hok/Sok system as the suicide switch for our engineered strain. In this system, the Hok protein functions as a pore-forming toxin that is constitutively expressed at a basal level, inducing programmed cell death in bacteria. Meanwhile, Sok is a small regulatory RNA whose transcription is activated by bile salts. Once expressed, Sok binds to hok mRNA and inhibits its translation, thereby neutralizing Hok's basal toxicity.

In the intestinal environment, bile salts induce the expression of Sok, maintaining a dynamic balance between Hok and Sok so that the engineered strain can survive normally. However, once the bacteria leave the intestinal environment, the absence of bile salts prevents Sok expression, leading to unrestrained Hok activity and triggering self-elimination of the engineered cells. This design enables precise responsiveness to environmental signals, providing a reliable and controllable biocontainment mechanism for engineered probiotics.

3. Design

We chose the plasmid backbone pIP501, a low-copy-number and strictly regulated plasmid controlled by the RepR replicon, known for its stable inheritance in Lactobacillus casei. In our design, the hok gene is placed downstream of the preca promoter, while the sok sequence, driven by the P16090 promoter, is inserted in the reverse orientation to establish the toxin–antitoxin system.

Specifically, when the engineered strain is cultured in bile-salt-containing medium or colonizes the intestinal environment, both Hok and Sok are expressed simultaneously. The Sok mRNA hybridizes with hok mRNA, suppressing its translation and preventing activation of the suicide system. However, once the engineered bacteria leave the gut environment, sok transcription ceases. The unopposed Hok protein forms pores in the bacterial membrane, leading to controlled cell death of the engineered strain.

4. Organism

The chassis microorganism used in this project is Lactobacillus casei, a species widely recognized as safe and extensively used in the food industry and probiotic products. It poses extremely low risk to both humans and the environment. The choice of L. casei is based on its excellent compatibility with our target application environment: as a natural commensal of the human gut, it possesses innate advantages in colonization and metabolic adaptation within the intestinal tract.

More importantly, such colonization is transient — the engineered strain is naturally excreted through the digestive process rather than permanently colonizing the host. This transient behavior minimizes the ecological risk of disrupting gut microbial balance or causing long-term environmental dissemination, ensuring biosafety from both ecological and health perspectives.

5. Conditions

Our experiments were conducted under certified and secure laboratory conditions to ensure both the success and safety of all procedures. We operate a standard Biosafety Level 1 (BSL-1) microbiology laboratory, which is specifically designed for low-risk microorganisms such as Escherichia coli and Lactobacillus casei. The laboratory is equipped with an ultra-clean workstation, biosafety cabinets, and essential protective equipment including disposable medical gloves and masks.

In addition to the basic facilities, we have also established a semi-enclosed laboratory that meets higher standards for aseptic and safety requirements. This area is equipped with professional biosafety cabinets designed for more delicate or sterile operations. To strengthen personal protection, our laboratory provides all members with sufficient supplies of disposable gloves and masks to significantly reduce the risk of inhalation or direct contact with microorganisms.

Clear and visible safety signs are placed at the entrance and within the laboratory to remind every member of safety regulations and operational precautions. These visual warnings ensure that everyone working in the facility remains aware of biosafety requirements at all times.

Figure.1 BSL-1 Laboratory

Figure.2 Cell culture chamber

Figure.3 Biological safety cabinet

Figure.4 Anaerobic Workstation

Figure.5 Biochemical incubator

6. Risk Management

Our laboratory has established a comprehensive risk management system to ensure a safe working environment for all personnel. Emergency medical kits are available for immediate use in case of accidental exposure or injury. Safety guidelines and standard operation protocols are prominently displayed on the walls to continually reinforce safety awareness among all team members. Emergency contact information and safety office hotlines are also clearly posted near entrances and exits, allowing rapid access to help if needed.

To further protect laboratory personnel, we maintain strict access control. Entry to the entire laboratory building is regulated through an activated campus card system. Only individuals who have completed safety training and obtained approval from the university's Laboratory Safety Center are authorized to access the facilities. Each specific laboratory room key is managed by designated personnel, ensuring that only authorized team members can enter.

In addition, we have implemented a standardized management system for hazardous chemicals and biological materials. These substances are stored in clearly labeled safety cabinets marked with warning symbols. Keys to these cabinets are strictly managed by responsible personnel, ensuring that access to potentially hazardous materials is carefully monitored and recorded.

7. Laboratory Access Security

Strict access policies are enforced throughout the entire laboratory complex. Individuals seeking entry must use their activated campus cards; unauthorized cards will not grant access. Only those who have completed the required training and received approval from the Laboratory Safety Center are eligible for card activation. Additionally, keys to specific laboratory areas are held by designated staff, ensuring that only approved members may enter. This system guarantees a controlled, secure environment for all research activities.

8. Experimental Training

Before performing any experiment, every team member undergoes comprehensive laboratory safety and technical training. The training covers a wide range of essential skills, including bacterial inoculation techniques, correct sterilization procedures, waste management, and proper use of personal protective equipment (PPE). These preparations significantly reduce risks associated with microorganism exposure.

At the end of the training program, supervisors conduct evaluations to assess each member's proficiency. Only those who demonstrate full competence are granted access to laboratory operations. Team members who do not meet the safety or skill requirements must continue training until they are qualified. This system ensures that every person working in the lab is both capable and responsible.

9. Waste Treatment

All waste generated from our experiments is handled following the biosafety guidelines outlined in the Biosafety in Microbiological and Biomedical Laboratories (BMBL) and according to the waste management standards established by Lanzhou University. Experimental waste is carefully classified into biological, chemical, and sharps categories. Each category is collected in designated containers that are clearly labeled with hazard symbols.

Autoclaving is performed at 121°C for at least 30 minutes to sterilize all microbial waste before disposal. Chemical waste and ethidium bromide-contaminated liquids are collected separately in marked containers and transferred to the university's centralized hazardous waste station at scheduled intervals, where they are processed by trained personnel. Sharps such as needles and glass slides are placed in puncture-resistant containers to prevent injuries.

10. Opinion Acquisition

To ensure comprehensive safety management, we actively seek advice and feedback from experts, professors, and biosafety officers. During the project, we presented our design to several biosafety specialists, who provided constructive suggestions on laboratory procedures, containment strategies, and potential risk control.

We also communicate with the public to introduce our project and gather their opinions and concerns regarding biosafety. By monitoring and addressing these safety-related questions, we continuously refine our project design to minimize risk and enhance public confidence.

11. Guideline

All of our laboratory operations strictly comply with relevant national laws and institutional policies. Specifically, in China, we adhere to the following key regulations and standards:

• Law of the People's Republic of China on the Prevention and Control of Infectious Diseases
• Regulations on Biosafety Management of Pathogenic Microbiology Laboratories
• Regulations on the Management of Medical Wastes
• General Requirements for Laboratory Biosafety (GB19489-2008)
• General Biosafety Standards for Microbiological and Biomedical Laboratories

At Lanzhou University, all experiments are performed under the supervision of authorized instructors and within the biosafety framework established by the School of Life Sciences. Additionally, we follow the specific safety requirements of other collaborating laboratories to ensure full compliance with both institutional and national biosafety standards.