Biosafety was introduced in response to the risks posed by biotechnology, particularly the potential for genetically modified organisms (GMOs) or their genes to escape into the environment. It involves measures to prevent gene leakage, which can harm ecosystems and human health. Preventing the unintended release of GMOs is essential to protect biodiversity, ensure ecological balance, and maintain food security. By controlling gene leakage, biosafety ensures that biotechnology advancements do not pose unforeseen risks to both society and the environment.

Our final application of kaempferol is as a food additive. If a standard strain of Escherichia coli (E. coli ) is used as the chassis microorganism for producing kaempferol, endotoxins generated during food production could cause consumers to develop foodborne illnesses, such as abdominal cramps and diarrhea. Therefore, when selecting a chassis microorganism, we prioritize safety and efficiency.

E. coli Nissle 1917 (EcN) is a non-pathogenic strain of E. coli , originally isolated in 1917. It does not contain pathogenic factors nor does it produce cytotoxins.It does not contain pathogenic factors and does not produce any cytotoxins. As one of the few Gram-negative bacteria used as a probiotic, EcN is widely applied clinically for preventing infectious diarrhea, treating inflammatory bowel diseases like ulcerative colitis and Crohn's disease, preventing the colonization of pathogens in the digestive tract of newborns, and supporting immune modulation.

Research has demonstrated that EcN is suitable for clinical medicine development and food production. Therefore, we chose EcN 1917 as our chassis microorganism to ensure that the kaempferol we produce does not contain endotoxins, guaranteeing the product's biosafety. This not only helps prevent foodborne illnesses but also makes our product more advantageous and reliable as a food additive.

We have chosen GAD, FLS, F3H as the fundamental genes of our products. Our study showed that the expression levels of these three genes were stable, with no toxic agents, no long-term side effects, and no environmental impact. The reasons are as follows:1. These genes may have a relatively stable genetic background in organisms, so that their expression levels can remain relatively stable under different conditions. 2. Since the biological function of these genes is not directly related to the production of toxin factors, they do not produce toxin factors. The production of toxin factors in organisms is usually controlled by other specific genes and regulatory mechanisms that differ in function and regulation from the genes mentioned above. Therefore, they are suitable for use as gene fragments. In addition, we have observed that these three genes have been widely used in experiments and no safety concerns have been reported related to their use.

Substrate: naringin, glycine

Products: kaempferol, GABA

Naringenin is a flavonoid compound commonly found in grapefruit and citrus fruits such as oranges and lemons. It belongs to the flavonoid family and has antioxidant properties, which can neutralize free radicals, potentially slowing the aging process and reducing the risk of chronic diseases. Naringenin also helps reduce inflammation in the body and may have a positive effect on inflammatory conditions such as arthritis. Consuming naringenin from grapefruit and other citrus fruits is generally safe for most people and does not cause adverse effects. As a supplement, naringenin is also usually safe when taken at recommended doses.

Glycine is an amino acid and an essential component in protein synthesis in the body. It is the simplest amino acid, found in various foods, including meat, fish, beans, and dairy products. Glycine is a key ingredient in collagen synthesis, which is crucial for the health of skin, bones, and joints. It has neuroprotective effects and may help improve sleep quality, cognitive function, and alleviate anxiety and depression symptoms. As an amino acid, glycine is widely found in food and is generally safe. However, high doses may cause gastrointestinal discomfort, such as diarrhea, nausea, or stomach pain, although these issues are uncommon at normal doses.

Kaempferol is a naturally occurring flavonoid, part of the flavonoid compound group, and is widely found in many plants, including green tea, spinach, apples, and grapes. Kaempferol is beneficial for the cardiovascular system, potentially helping to lower blood pressure and improve blood lipid levels, thus reducing the risk of cardiovascular diseases. It may also have neuroprotective effects, improving cognitive function and helping prevent neurodegenerative diseases. As a plant-based compound, kaempferol is generally safe, and most people do not experience adverse reactions when consuming kaempferol-rich foods. However, a small number of individuals may be allergic to flavonoids in certain plants, potentially triggering allergic reactions.

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter that plays an important role in the central nervous system. It helps regulate nerve activity and has calming and relaxing effects. GABA has sedative properties, helping to relieve anxiety, stress, and insomnia, and is commonly used as a supplement to improve sleep quality by aiding in falling asleep and maintaining deep sleep. GABA may also protect the nervous system and slow the progression of neurodegenerative diseases. Naturally present in the body as a neurotransmitter, GABA is safe when consumed through food. GABA supplements are generally considered safe when taken at recommended doses.

In this project, we need to ferment our engineered bacteria in a factory setting, then proceed with cell disruption and purification to extract the useful components. Therefore, it is crucial to determine how to effectively disrupt the cells and ensure that all bacteria are completely inactivated after fermentation. We conducted research on this matter.

In laboratory settings, the traditional method for inactivation is autoclaving, while filtration can be used for smaller volumes. However, at the factory scale, we found that ultrasonic disruption is the most suitable method for breaking down the cells. Although ultrasonic disruption effectively destroys the cell structure, we are concerned that some bacteria may survive. To address this, we propose using filtration sterilization after ultrasonic disruption to ensure that the final filtrate contains no viable engineered bacteria, thus preventing any gene leakage or residual live bacteria.

In the laboratory, autoclaving is commonly used to treat most solutions, utilizing high temperatures and pressures to kill microorganisms and ensure sterility. While this method is effective, for certain heat-sensitive components, autoclaving may cause degradation. In such cases, we recommend autoclaving the other components first, and then adding the heat-sensitive components, which have been sterilized via filtration, after the solution has cooled to room temperature. This ensures both sterility and the integrity of the solution.

Choosing the appropriate method of inactivation is not only crucial for the success of the experiment but also for ensuring safety throughout the process.

Before entering the laboratory, we underwent comprehensive and thorough safety training. This training covered a wide range of topics, including access control, proper handling of laboratory waste (solid, liquid, and gas), safety protocols for water, fire, electricity, and gas, as well as the safe use of flammable, explosive, and corrosive reagents. It also included detailed guidance on the classification and disposal of laboratory waste. Moreover, special attention was given to the proper selection and use of personal protective equipment (PPE), such as gloves, respiratory protection, and eye protection. These measures ensured our safety during lab operations and helped mitigate potential risks and hazards.

Conduct experiments under the supervision of teachers, wear lab coats and gloves, properly dispose of experimental waste, and do not smoke or eat in the laboratory.

When entering the laboratory, wear lab coats and disposable gloves and masks, do not bring food or water into the laboratory for eating, and do not smoke in the laboratory.

As a student, you must conduct safe experiments under the supervision of laboratory teachers, listen carefully to the teacher's experimental explanations, avoid contamination of experimental products caused by improper operation during the operation, and use more dangerous laboratory items with caution, avoid contact with skin and desktop, and avoid accidental ingestion, and properly dispose of experimental waste after completing the experiment. Take off gloves and lab coats and wash your hands before leaving the laboratory.

Our project aims to find a new method to alleviate emotions such as depression and anxiety, while prioritizing safety throughout the experimental process. We selected the non-pathogenic E. coli Nissle 1917 as our chassis microorganism to ensure that no harmful substances are produced during the process. Before using the strain, it was thoroughly sterilized to prevent any risk of infection. The substrate we used is a natural plant that is safe and widely applied in the pharmaceutical and food industries. The final products, kaempferol and GABA, are both verified safe components. The entire experiment was conducted in strict accordance with biosafety regulations, under sterile conditions, and all procedures were safely completed with the supervision of trained personnel. We also consulted authoritative experts to ensure that the project complied with relevant laws and regulations while fully guaranteeing the biosafety of the final product.