As synthetic biology and gut microbiota research continue to advance, there is a growing number of synthetic biology projects focusing on gut microbiota. However, an inevitable challenge faced by all teams working with gut microbiota is how to ensure biosafety. After all, if genetically modified chassis microorganisms are released into the environment through fecal discharge following gene modification, it would be an environmentally irresponsible act.

Currently, the majority of teams are employing methods that involve the insertion of various suicide genes into chassis microorganisms, along with the use of different specific promoters to induce suicide behavior under different conditions. However, all of these methods have two fatal shortcomings. Firstly, suicide genes can pose varying degrees of harm to the host organism and potentially to humans. Secondly, these suicide systems cannot guarantee 100% cell death, leading to a high risk of leakage.

Considering the lack of effective biosafety measures for gut microbiota, our team suggests that many synthetic biology projects based on gut microbiota do not necessarily need to operate within the gut. They can be conducted in vitro, which can significantly mitigate potential biosafety concerns.

We also encourage many teams working with gut microbiota to refrain from blindly designing projects based on gut microbiota if safety cannot be assured. Instead, they can explore rational gene circuit designs conducted in vitro as a viable alternative.

1.Safe way to use

1) Analysis of legal structure

In an effort to ensure the feasibility and legitimacy of our project, we sought and scrutinized relevant legislation and protocols, thus eliminating further violations of ethics and security to a greater extent. Aside from pure scrutinization, the application of the legal provisions in our project is also taken into consideration, which aims to provide guidance on the proper implementation of our project.

One principal reason why we finally chose to design a specific kind of food additive as our probiotic product is the strict regulations present in our country, causing the unavailability of legal permissions to produce and promote probiotics that function in human intestines as a tool for lowering cholesterol levels. Involving posing a myriad of health risks to consumers that can violate ethics and biosafety, it is doubtless that the protocols on probiotics and drugs that function within human bodies are strictly enforced.

As illustrated in the PRC Convention on Biology Safety and PRC Pharmaceutical Administration Law, engaging in clinical research on biomedical technologies involving public health should rigidly adhere to the scientific ethical values and experiments should be conducted by certificated specialists in legal medical institutions that possess qualified conditions. Additionally, the medical institution should also implement filing management with regard to relevant departments, and the designed trial protocols should be examined and approved by the Biosafety Review Board. These rigorously enforced constraints indicate that if we decide to design probiotics that function within human bodies in which clinical trials are involved, legal permissions from relevant government departments, professional staff, equipment, and test sites must be obtained for security reasons before launching our project to start experiments, which are all rather difficult to access in a short space of time. Also, further promotions and issues on marketing would undergo further complex and cumbersome legal procedures. 

2) Safe way to use

Our original plan was to effectively control cholesterol levels throughout the human body by proposing the usage of probiotics by adopting microbial therapy in vivo to allow the probiotics function in human intestines. As our investigation continued, however, we discovered that there may be significant risks when E. coli bacteria is placed in human intestines after consulting relevant literature and professionals in the medical research field. More importantly, relevant laws and regulations in China have always been rigorously enforced, causing it to be rather difficult to get legal approval to put engineered probiotics that function directly in vivo in mass production. Therefore, we realised that producing powdered probiotic products in vitro is our best option, given its high efficiency in degrading cholesterol along with the absence of risks. After thorough investigation, we decided to produce food in factories with our bacterial powder as an additive to promote safety and efficacy of our product for our consumers.

2.###3) Selection of non-pathogenic strains：
In our experiments, we used BL21 and DH5-alpha Escherichia coli cells for transformation and cloning purposes. Both BL21 and DH5-alpha strains of E. coli are non-pathogenic and have been developed for laboratory cloning purposes. BL21 is one of the most commonly used strains for prokaryotic expression, known for its safety features of being non-toxic and non-pathogenic. BL21 is a proteinase-deficient B strain lacking the La chemoreceptor, which is why it does not produce toxins! DH5-alpha E. coli is also a safe, non-pathogenic strain with a higher growth rate and transformation efficiency, making it suitable for gene transformation experiments without posing a threat to the human body. DH5-alpha E. coli carries several important gene mutations, such as lacking restriction enzymes mcrA, mcrB, mrr, hsdRMS, which makes it easy to accept exogenous DNA. The potential health and environmental hazards associated with BL21 and DH5-alpha E. coli are extremely limited, and therefore, these strains can be handled in a biosafety level 1 laboratory. To minimize potential health and environmental hazards, laboratory personnel need to follow proper operating procedures, including wearing appropriate personal protective equipment and properly handling and disposing of waste. In case of release of E. coli in the environment, immediate cleanup and disinfection procedures is necessary.

2. Selecting genes which are harmless to the human body:

The three genes chosen by our team are the galU gene, ACS gene, and ismA gene.

1）Safety features of the galU gene:

The galU gene plays an important role in metabolism and encodes UDP-glucose pyrophosphorylase, which synthesizes UDP-glucose pyrophosphate as a component of cell walls, cell membranes, and intracellular carbohydrates. The galU gene is widely found in bacteria and is one of the key genes for bacterial membrane and biofilm synthesis. This gene has certain applications in drug design and medical research; for example, targeting the galU gene can be used to design drugs that inhibit bacterial biofilm formation for treating various infections and drug-resistant bacteria. 

2） Safety features of the ACS gene

The ACS gene is the gene encoding 1-aminocyclopropane-1-carboxylate synthase, which plays a crucial role in the metabolism of ketone fatty acids. Fatty acids are important building blocks of cell membranes and also serve as a source of energy in living organisms.

3） Safety features of the IsmA gene

In the human gut microbiome, the ismA gene is involved in cholesterol metabolism and is present in an uncultured bacterial lineage belonging to the clostridia cluster IV, commonly found in different populations across various geographical distributions. It encodes a microbial cholesterol dehydrogenase that contributes to the formation of coprostanol. Individuals which have coprostanol-forming bacteria in their bodies exhibit significantly lower serum total cholesterol levels, comparable to changes in lipid homeostasis genes.

3. Characteristics and safety features of Escherichia coli 1917:

After determining the form in which our probiotics would be produced, we needed to find a reliable and suitable carrier. After discussion, we decided to choose E. coli as our chassis, which is already found in human intestines. Most types of E. coli possess pathogenicity and can create risks to human health in a myriad of aspects. However, we must seek a certain type of carrier that is absent of pathogenicity for security reasons. Therefore, following further discussion, we decided to use the bacterial strain E. coli Nissle 1917, given its quality biocompatibility, ideal targeting ability, and most importantly, its absence of pathogenicity. 

We used the E. coli Nissle 1917 (ECN) as a carrier of our probiotic product which may function unitedly in vivo following the genetic circuit we structured with the T7 promoter, a specific kind of cholesterol ester transfer protein, the oleic acid promoter, the ACS gene (which encodes the ACS, also known as the acyl-CoA synthetase), and the IsmA gene (which encodes a kind of HSD, also known as the hydroxysteroid dehydrogenase). With a complete and reliable genetic circuit, our product can be safe to consume.

As one of the most widely used engineering bacteria in nano drug delivery systems, E. coli Nissle 1917 (ECN) displays high performance in practical use. Compared with other engineering bacteria, ECN possesses impressive antibacterial, anti-inflammatory, regulating intestinal microflora and facultative anaerobic characteristics. Apart from promoting intestinal peristalsis, ECN can also effectively maintain the stability of the intestinal flora by inhibiting the colonization of pathogenic bacteria and the production of toxins. Additionally, ECN avoids inflammation caused by mucosal bacterial attachment and invasion to some extent. To conclude, ECN plays a crucial and positive role in repairing first-line immunity and regulating innate immunity. Therefore, ECN can provide a potent guarantee for the security and innocuousness of our product, further ensuring the user‘s physical well-being.

4. Solutions to biosecurity problems that may be faced in real-world environments

1）Using a filter:

Installing a filter on the container can effectively prevent bacteria from entering or leaving the container. This filter typically has micropore sizes which block the passage of bacteria while allowing the free flow of gases.

In order to reduce the levels of cholesterol often found in dairy products, such as eggs, a sterilization instrument can be designed by creating a membrane filtration technology which can then be integrated with the bacterial powder treatment process. This instrument can directly filter the bacteria added during the food processing process. The mixing liquid undergoes separation through a membrane system, with sufficiently small pore size to effectively remove solid residues from bacteria and bacterial powder. The preliminary filtered dairy products flow out from the outlet of the instrument and undergo another low-temperature sterilization simultaneously. As a result, the bacterial and solid residues are left completely in the filter membrane system or killed, and the factory can clean or replace the filter membrane regularly. By using this sterilizer, factories can effectively prevent bacterial leakage and avoid food safety issues.

2）Ultraviolet disinfection:

Settng up ultraviolet lights inside the container can kill or 
prevent the growth and reproduction of bacteria through ultraviolet radiation. UV 
lamps are usually placed on the top or sides of the container to ensure that the 
radiation reaches the entire interior of the container.（

3）Temperature control:

The growth of bacteria in an effective manner usually requires a suitable temperature range. By controlling the temperature in the container, the reproduc-on of bacteria can be limited. Maintaining extremely low or high temperatures, or using thermostatic equipment to control the temperature, can 
effectively inhibit the growth of bacteria.

4）Material selection:

Choosing materials with good an-bacterial properties as the 
construction material of the container can reduce the growth of bacteria in the 
container. For example, the use of antibacterial materials such as copper and 
stainless steel can effectively inhibit the attachment and growth of bacteria.

5）Protective layer:

Coating the surface of the container with an antibacterial coating or adding antibacterial agents into the container can form a protective layer to prevent bacteria from multiplying in the container. These protective layers work by killing bacteria or preventing them from adhering to container surfaces

5. Safety of the final product

The main ingredients in our product are not significantly different from those found in normal eggs and milk. Egg products are mainly consist of raw eggs, and the materials of dairy products are also made from conventional raw milk. However, in the production of these eggs and milk products, we will add our bacterial powder to lower the cholesterol in food. When engineering bacteria completes the degradation of cholesterol in food raw materials, we will sterilize these semi-finished products with added bacterial powder to avoid the leakage of our engineered bacteria to ensure that these products may be safely consumed. Finally, because the food has undergone sterilization before leaving the factory, our product will not produce any harmful substances during the natural metabolic process of the human body after consumption. The metabolites will be consistent with normal egg milk metabolites.

6. Survey of public acceptance of cholesterol-lowering foods

1. Safety training

1) Online training：

Before starting to perform experiments in the laboratory, all members received comprehensive training on laboratory etiquette and safety protocols through our online meetings. Emphasis was placed on practices such as wearing proper laboratory attire and protective gloves when handling chemicals to minimize the risk of exposure to harmful substances, and ensuring a safer and more sterile working environment.

2) Safety laboratory quiz：

We created a safety laboratory quiz, requiring members to score above 95% to earn a pass to enter the laboratory and perform relevant experiments, ensuring that all members are familiar with the meaning of various safety symbols in the laboratory and the safety laboratory rules that have to be followed.

3) Laboratory guidance teacher：

Our laboratory guidance teacher is a graduate student from Shanghai Jiao Tong University. He supervised us while we performed experiments to ensure that we took the necessary precautions, strictly followed the experimental instructions, and avoided potential accidents. This allowed for the smooth progression of the experiments.

2. Innovative laboratory safety management

1) Laboratory rules

Lab personnel are required to dress neatly and are strictly prohibited from wearing slippers or tank tops, and long hair should be neatly tied. The laboratory should be kept quiet, clean, and hygienic during experiments. After preparing for the experiment, strict adherence to experimental requirements, careful observation, in-depth analysis, and accurate recording should be followed. Experiments should be completed on time with high quality. Lab personnel should take care of the equipment, conserve water, electricity, and consumables, and should not use any equipment or items unrelated to the experiment without permission, nor should any laboratory items be taken outside of the laboratory area. Safety and precaution should be exercised during experiments, and in case of accidents, the power supply should be shut off immediatly. After completing the experiment, the power supply, water, and gas should be promptly shut off, and the used equipment should be put back to their relevant cabinent/freezer. Experimental reports should be written diligently and promptly according to the experiment requirements, analyzing experimental results and accurately processing experimental data without altering the original data. When conducting self-designed experiments in the laboratory, prior contact with the relevant laboratory should be made to report the purpose, content, and required experimental equipment and materials.

2) Personal requirements

Eating, playing, and smoking are strictly prohibited in the laboratory, and items unrelated to the laboratory and experiments should not be brought into the laboratory. Protective equipment should always be worn when performing experiments, and equipment should be checked regularly for any defects and damage. If any defects are found, the issue should be reported to the management team and equipment should be repaired or replaced as soon as possible. Every lab member should be well versed in the emergency response procedures. Experiments should not be left unattended, and permission from the teacher or primary researcher should be obtained for dangerous experiments. Safety measures such as water, electricity, and fire safety should be taken, and safety agreements should be signed when purchasing products from external companies to avoid negligence in the purchased products. The condition of all safety equipment, including fire extinguishers and emergency showers, should be checked up regularly. Personal protective equipment, including laboratory coats, goggles, gloves, and masks, must be worn according to experimental standards to ensure protection against chemicals, biological hazards, and other potential dangers. Experimental waste and equipment debris should be collected separately and properly disposed of according to the relevant regulations. Familiarity with the meanings of various laboratory warning signs and memorization of emergency response procedures for laboratory safety incidents are necessary. The laboratory should also pay attention to safety management and inspections related to water, electricity, and gas. Before leaving the laboratory, water, electricity, gas, doors, and windows should be properly shut off.

3) Equipment usage requirements：

Regular cleaning and calibration of equipment should be conducted, and damaged equipment should be fixed or replaced promptly. In case of equipment malfunction, remarks should be added promptly to avoid further damages or impact on experimental accuracy. To improve experimental accuracy and efficiency, comprehensive usage guidelines should be established, including experimental conditions, operating procedures, and data analysis methods. Additionally, registration should be done promptly after using instruments.

4) Waste disposal：

Laboratory-generated waste should be categorized with appropriate labeling, undergo disinfectoin, and then be stored properly.

Waste grade:

5) Zoning

The laboratory should be properly laid out, including appropriate placement of equipment, ensuring cleanliness of corridors without clutter, and separating experimental areas from study and office areas. When performing our experiments, we implemented zoning within the laboratory to prevent cross-contamination since we were performing different experiments simultaneously. Zoning experiments meant we were able to effectively improve experimental efficiency and ensured the swift progress in completing our required experiments.