SAFETY

Safety and security rank as the most crucial principle among all aspects of experimentation. The safety regulations of HainanU-China have been meticulously formulated to guarantee that all members are adequately protected during the process of experiments. During the CCiC, our team discussed the safety issues that need attention throughout the iGEM competition together with teams such as CSU-CHINA and NKU-China. Mutual safety inspections were conducted among the teams, and subsequently, we also carried out the following self-inspection of our own safety based on the content of the meeting. Both our wet-lab and dry-lab experiments are designed with strict adherence to safety norms in every aspect. All members involved in the experiments have successfully completed rigorous laboratory access training and assessment programs conducted by Hainan University. Our laboratory is classified as a BSL-3 facility. Throughout the entire experimental process, strict compliance is maintained with regard to the relevant specifications for biological materials and chemical reagents. Under any circumstance no one can these regulations be violated.

Before the experimental activities were launched, the student leaders strictly required all the experimental members to pass the laboratory safety examination and obtain the Laboratory Safety Access Certificate of Hainan University as a necessary condition for entering the laboratory to carry out the project. All members of HainanU-China spent two weeks learning about the instruments and consumables involved in the experiment and practiced operating them under the guidance of the PIs until they could use them proficiently. We have also completed the writing of an instrument safety document, hoping to provide assistance and guidance for the safety activities of the iGEM community. Each laboratory member has a copy of the Laboratory Safety Manual published by the Laboratory and Equipment Management Office of Hainan University, which contains the following contents:

• Laboratory Safety Rules
• Safety of Hazardous Chemicals
• Fire Safety
• Safety of Instrument and Equipment Use
• Radiation Safety
• Laser Safety
• Biosafety
• Emergency Response to Laboratory Accidents
• School Laboratory Safety Management Methods

All team members have memorized these contents by heart, which is the greatest guarantee for the safe conduct of our experiments. At the same time, this manual is also placed in the laboratory for reference when needed.

Fig 1. The main contents of the Laboratory Safety Manual Fig 2. Laboratory Safety Access Certificates of Experimental Team Members

On the morning of August 8th during the CCiC, our team jointly organized an iGEM-related exchange meeting focusing on biosafety and ethical issues in collaboration with representatives from CSU-CHINA、NKU-China、Fudan, and other institutions.

During the meeting, we interpreted iGEM’s requirements regarding biosafety and ethics related to social practice, summarized the biosafety work experience of outstanding teams in previous years, and each team separately introduced their projects, raised ethical and biosafety concerns encountered during the competition, and worked together to resolve these issues.

We collectively read and summarized the Biosafety Law of the People's Republic of China (Amended in 2024), gaining clarity on the safety issues that require attention throughout the competition. Central South University shared the Ethical Review Measures for Life Science and Medical Research Involving Humans, covering aspects such as informed consent, information disclosure, and risk prediction.

After this meeting, based on the suggestions provided by other teams and relevant reference requirements, we conducted a self-inspection of our project’s safety—including safety checks on chassis microorganisms and biological parts. Additionally, we installed a suicide switch to further enhance the safety of our product.

Fig 3. Mutual Safety Inspections Among Teams at the Roundtable Meeting

Bacteria Strains

In the design and implementation of the experiment, we mainly dealt with two different strains of Escherichia coli: E.coli DH5α and E.coli Nissle 1917. E.coli DH5α served as the plasmid cloning vector for the prokaryotic biomass, playing a significant role in plasmid construction and amplification; E.coli Nissle 1917 was the chassis strain of the engineered bacteria, using glucose as the substrate to undergo a series of metabolic reactions to ultimately obtain the BHB required for the project.

E.coli DH5α is regarded as one of the safest and most widely used strains in biological laboratories, belonging to the biosafety level BSL-1 microorganism. This means that under normal laboratory operation conditions, it does not pose a pathogenic threat to healthy adults. This strain has the following core safety characteristics:

Non-pathogenicity: E.coli DH5α originated from the Escherichia coli K-12 strain. The K-12 strain is a model organism that has been cultivated and modified in laboratories for decades, and its key pathogenic genes have been removed or inactivated. It cannot effectively colonize and survive in the intestines of healthy humans or animals, thus not causing diseases.

·Genetic defect type: E.coli DH5α has several key nutritional defect types and gene mutations, which are both advantages as a cloning vector and enhance its safety:

recA1 mutation: Missing the DNA homologous recombination repair function. This makes exogenous DNA more stable in the host and less prone to recombination, and also significantly reduces its ability to exchange genes with other bacteria.

endA1 mutation: Missing the exonuclease I, which is conducive to extracting high-quality plasmid DNA.

hsdR17 (rK-, mK+): Missing the restriction endonuclease system (R-M system) of the restriction enzyme (R), but retaining the methylase (M). This makes it not degrade exogenous DNA, but at the same time limits its ability to defend against foreign DNA, making it extremely weak in competitiveness in the natural environment.

Dependence on laboratory environment: Due to the above genetic defects, E.coli DH5α is a very weakly viable strain. It requires a rich LB medium and a specific 37°C growth temperature. Once it leaves the laboratory environment, it is difficult to survive in nature or in the host.

E.coli Nissle 1917 is a well-studied probiotic strain that is safe for oral use in healthy individuals. It has been used safely for decades as a medicine and dietary supplement globally (especially in Europe). This strain has the following core safety characteristics:

·No key virulence factors: This is the fundamental safety of E.coli Nissle 1917. Genome sequencing confirmed that E.coli Nissle 1917 lacks all known major pathogenic virulence genes, such as alpha-hemolysin, Shiga-like toxin, pathogenic island, cell toxin necrotic factor, etc. It retains some iron carrier systems and other metabolic-related factors, which instead help it compete with harmful bacteria in the intestine and exert a probiotic effect.

\

·Long-term human use history: E.coli Nissle 1917 was discovered in 1917 and has been made into a medicine for clinical use for decades, used for the prevention and treatment of various intestinal diseases, accumulating a large amount of safety data.

·Good biological characteristics: E.coli Nissle 1917 can effectively colonize in the human intestine and inhibit the growth of pathogens such as Salmonella and Shigella through competition for nutrients and ecological niches, thereby protecting the host. Studies have shown that its genome is relatively stable and is not prone to acquire or transfer virulence genes.

Although these two strains have been proven to be safe, when handling any microorganisms in the laboratory, good microbiological operation protocols (GMP) and safety regulations must be followed:

• Personal protection: Wear laboratory coats, gloves and goggles.
• Aseptic operation: Perform operations near an alcohol lamp or a biosafety cabinet to reduce contamination and aerosol generation.
• Disinfection: All items that have come into contact with the bacteria must be subjected to high-pressure steam sterilization (121°C, at least 20 minutes) before disposal. The work surface should be wiped with 75% alcohol or bleach after the experiment.
• Prohibition: Do not eat, smoke, store food, or touch your face with your hands in the laboratory.

Parts Safety

Apart from the strains, we also rigorously reviewed all the organisms, parts involved in the project. All of them come from the iGEM White List and have excellent biological safety guarantees. In terms of the dry experiments, the hardware cEEGrid uses anti-static shell materials, and the electrode material is a flexible electrode, which largely ensures safety. In addition, we also selected advanced Bluetooth to avoid potential information loss and leakage risks during Bluetooth transmission.

Table 1.The gene parts used in the experiment and their corresponding sources

Gene	Source
pcT	Megasphaera elsdenii
phaA	Ralstonia eutropha
phaB	Ralstonia eutropha
rplO	Escherichia coli str. K-12 substr. MG1655

Design

Hydrogel Barrier

The intestinal environment where the engineered bacterium E.coli Nissle 1917 colonizes harbors an extremely rich intestinal microbiota, consisting of a variety of different intestinal bacteria. Among these bacteria, there are extensive contacts and interactions, resulting in highly active horizontal genetic material exchange. If E.coli Nissle 1917 is completely exposed to the microenvironment of the intestinal microbiota, the exogenously introduced plasmids and genes are at a considerable risk of being lost or exchanged, thereby potentially inducing immeasurable disruption to biological safety.

To address the abovementioned issues, we designed an experimental protocol involving the encapsulation of E.coli Nissle 1917 in a hydrogel. Hydrogels are a class of hydrophilic polymeric materials with a three - dimensional network structure. Leveraging their unique stability, a physical barrier can be effectively established to isolate potential contacts between E.coli Nissle 1917 and intestinal bacteria, thus avoiding safety issues such as genetic material loss and gene leakage.

Simultaneously, with the aim of achieving better colonization in the small intestine, the outer layer of the hydrogel is made of pH - responsive sodium alginate. This outer layer remains stable within the acidic digestive tract prior to reaching the small intestine. Once it reaches the alkaline environment of the small intestine, the outer layer of the hydrogel degrades, exposing the adhesive middle layer, which then physically adheres to the target site. In this manner, the hydrogel design not only alleviates concerns regarding potential gene leakage but also prevents the engineered bacterium from leaking into the environment due to unsuccessful colonization, thereby averting biological contamination.

Suicide Circuit

Although the above design has taken microbial safety comprehensively into account, and E.coli Nissle 1917 has been widely demonstrated to be a non - pathogenic probiotic, since the engineered bacterium carries genetically modified recombinant plasmids, there is still a possibility of causing incalculable damage to the environment. Therefore, it is essential to design a suicide circuit.It should be noted that the purpose of designing such a suicide circuit is to mitigate the problem of unforeseen gene pollution caused by the release and spread of the engineered bacterium into the environment due to the failure of the hydrogel to adhere to the small intestine. Compared with the external environment, one of the most distinctive environmental factors in the small intestine is the extremely low oxygen concentration. Based on this, we designed an oxygen - concentration - responsive suicide circuit to ensure that the engineered bacterium initiates the suicide process only when it enters the high - oxygen external environment after passing through the low - oxygen environment of the small intestine.

Before E.coli Nissle 1917 enters the low - oxygen environment of the intestine, the repressor - regulated promoter PhlF promotes the expression of the Cl repressor with an ssrA - tag. This Cl repressor specifically binds to the OR operator, preventing the micro - aerobic promoter PVHb from activating and initiating the suicide pathway. After ingestion and entry into the micro - aerobic environment, the hypoxia - responsive promoter PfdhF is activated, triggering the expression of the PhlF repressor protein. This protein then binds specifically to PhlF and inhibits its function, thereby shutting down the expression of the CI repressor. The ssrA - tag ensures that once the induction signal ceases, any pre - existing CI molecules within the cell are rapidly degraded. Consequently, the inhibition of PVHb is relieved.Subsequently, if the engineered bacterium is re - exposed to a high - oxygen concentration environment, PVHb will activate the expression of gene ccdB, leading to the suicide of the engineered bacterium.

Figure 4.Suicide circuit

Synthetic biology research is always confronted with ethical and safety considerations. Our project is closely connected with society in every aspect. It is our unshirkable responsibility and commitment to take ethical and safety as one of the highest principles in all activities. As none of our team members and PIs have a background in bioethics and safety research, we consulted Professor Cheng Guobin from Southeast University, an expert in bioethics, to seek advice on the ethical and moral evaluation and suggestions for all aspects of our project. Regarding the experimental design, he pointed out that our method of encapsulating live bacteria in hydrogels effectively ensures drug safety and has high drug accessibility. In terms of ethics, he suggested that we strictly adhere to informed consent and privacy protection in all HP activities. In fact, throughout the process of our HP activities, from design to implementation and data collection, we have strictly followed the principles of informed consent and privacy protection for all participants. For instance, when conducting interviews with patients and their family members, we designed an informed consent form and asked them to fill it out. All content related to the questionnaires was kept anonymous to protect the public’s privacy, truly fulfilling strict adherence to ethical principles.

Fig 5.Record of communication with Professor Cheng Guobin

Similar to safety, ensuring security is essential for the implementation of experiments. To safeguard the safety of laboratory personnel and the secure progression of experiments, we engaged with the laboratory management instructor for in - depth learning.

We discovered that the laboratory adheres to rigorous safety management systems. This encompasses pre - experiment safety training, guidelines for the proper use of chemicals, and specifications regarding personal attire.

Attire Requirements

During the experimental process, it is mandatory to don protective gear such as laboratory coats, rubber gloves, and safety goggles. Particular care should be taken when coming into contact with chemical reagents and biological materials, with a focus on avoiding any contact with hazardous substances.

Waste Disposal

All laboratory waste is meticulously sorted in strict accordance with laboratory safety standards. Additionally, comprehensive subsequent treatment measures are implemented to prevent any adverse impacts on the environment.

Emergency Protocols

The laboratory is outfitted with a complete set of emergency response equipment, including eyewash stations and fire extinguishers. This enables laboratory members to respond promptly in the event of an emergency.

By strictly regulating security aspects, we have not only heightened the awareness of experimental safety among team members but also effectively incorporated a series of measures into laboratory management. As a result, the entire project can be carried out within a safe and rational scope.