In today's world, the rapid development of biotechnology brings many opportunities, but it also comes with certain risks, especially in terms of biosafety. What is biosafety? The World Health Organization (WHO) has a definition of biosafety. A critical aspect of biosafety is the prevention of genetic leakage. Focusing on biosafety can protect human health, prevent biological invasion and species extinction, promote biological diversity and maintain ecological balance. Such as careless attention to safety measures or lack of safety awareness may be a threat to the environment and human life. Therefore, our team is fully aware of the importance of biosafety during the project, complies with the safety regulations of iGEM competition, carefully evaluates the risks during the experiment, and manages any risks from the process of project design and experiment operation.

E. coli is divided into pathogenic E. coli and non-pathogenic E. coli, pathogenic E. coli can cause a variety of health safety. Severe cases can include food poisoning, diarrhea, and infections of the digestive tract, urinary tract, or many other parts of the body. In order to avoid these problems in our experimental design, we plan to use a non-pathogenic E. coli nissle 1917（EcN）. EcN is a recognized, good probiotic that can be taken orally, which means that it does not produce any endotoxins that cause harm to the human body.

To avoid genetic leakage, which can harm the environment and nearby people and animals, we’ve incorporated a suicide system into our engineered bacteria. We designed a photosensitive suicide system not only because it is convenient and cost-effective, but also because it can be integrated with photonic skin rejuvenation devices, an emerging form of medical beauty.

We introduced a light-sensitive promoter, pdown (blue-green light induction), into the engineered bacteria. This promoter controls the expression of the suicide gene mazF, which triggers bacterial cell death at wavelengths of around 400-500nm. Through this system, when exposed to the appropriate light, the engineered bacteria are effectively eliminated, ensuring safety by preventing their survival and potential genetic leakage.

The proteins produced by the genes we use are harmless to the environment. They do not produce virulence factors, do not impose a burden on the environment, and do not disrupt the local ecosystem.

In the production system of genistein, the three genes involved are IFS, CPR, and HID. These genes have been proven in multiple research articles to be feasible and safe, posing no risk to human health or the environment. They safely and reliably convert the substrate naringenin into the product genistein.

In the bacterial cellulose production system, four genes— acsA , acsB , acsC , and acsD —are widely used in industrial production and commercial applications. This broad usage demonstrates the safety of these genes. Therefore, no toxic or harmful substances are produced or discharged during bacterial cellulose production.

In the blue light-induced suicide system, pDawn contains a light-sensitive promoter that is activated under blue light. When exposed to blue light from a photorejuvenation device, it activates the MazF suicide system, ensuring that no toxic or harmful bacteria remain.

Substrate:

Naringenin, a natural organic compound, is abundant in citrus peels. It has strong pharmacological effects, including antibacterial, anti-inflammatory, and immunomodulatory properties. It is widely used in the treatment of bacterial infections, as well as in sedatives and anti-cancer drugs. This indicates that using naringenin as a substrate is very safe.

Product:

We use a biosynthetic method to produce genistein, which originates from soybeans.

Genistein:

Genistein is the main active compound in soy isoflavones and is the most effective functional component. It also possesses a variety of therapeutic properties. Through literature review, we found that genistein has already been applied in various medical fields, further proving that our product, genistein, is safe and reliable, with no negative impact on human health.

Bacterial Cellulose (BC):

Bacterial cellulose is the material used for our membrane. After reviewing numerous studies, we found that bacterial cellulose is widely recognized as an environmentally friendly and safe material for human use. Bacterial cellulose is a natural biomaterial with excellent biodegradability. When released into the environment, it can be broken down by microorganisms into harmless substances. Additionally, bacterial cellulose has good biocompatibility and does not trigger significant immune responses or toxicity when in contact with human tissues.

Photon Beauty Device:After applying the mask, wear the photon beauty device and select the blue-green light mode. Following gene editing and testing, the blue-green light from the photon beauty device can effectively activate the suicide system, preventing bacterial leakage.

Removing the Mask:After removing the mask, rinse any remaining serum from the skin with clean water to avoid clogging the pores, which could otherwise lead to skin issues such as inflammation.

Disposal of Used Masks:After removing the mask, dispose of it properly to prevent environmental impact. The mask can be wrapped in a tissue and then sorted and discarded according to local waste disposal regulations.

Mask Usage Frequency:It is not recommended to use more than one mask per day. Avoid excessive or frequent use of masks, as overuse can lead to overhydration, weakening the skin’s barrier function and making the skin more prone to issues.

Before entering the physical laboratory, we underwent systematic safety training.

This training included instructions on the safe use of various laboratory instruments and equipment, including but not limited to alcohol lamps, biosafety cabinets, autoclaves, and centrifuges.

When entering the laboratory, it is required to wear gloves and a lab coat as per safety regulations. For experiments that may pose a risk to the eyes, safety goggles or face shields must be worn. The gloves used must meet general technical requirements, including mechanical protection, chemical protection, heat and fire resistance, protection from ionizing radiation, and defense against low-temperature hazards.

Domestic waste and laboratory waste shall not be thrown into the same trash can, and need to be thrown into a separate trash can outside the laboratory.

Laboratory waste should be placed according to the acidity and alkalinity of organic and inorganic materials. The different drop barrels need to be clearly indicated, and ensure that the container is intact, and no leakage can occur.For strong acids and bases, strong oxidants, strong catalysts and other strong chemical reactions, reagents need to be diluted before being put into the corresponding drop point.

problems should be reported in time to repair shall not be stored in the oven, dry, baked organic matter. It is forbidden to smoke or use open flames in the laboratory, and it is forbidden to pull wires carelessly. Prohibit the use of electric furnaces, electric heaters and other electrical appliances with open flames when people want to leave, the water and electricity must be turned off.

The laboratory will store a variety of flammable and explosive reagents, including solids and liquids. We should pay extra attention to the safety of the use of these reagents. In addition, the E. coli we used in this project has the possibility of being eaten by mistake in the experiment, so in the laboratory, we should pay attention to not eating or drinking in the laboratory, and it is best not to play with mobile phones.

In the laboratory, no one can perform experiments and work alone, and at least two people are required in the laboratory. This can effectively avoid the occurrence of danger. In the process of the experiment, one person can not find the possible dangers around in time, so the presence of the second person can guarantee the safety of the experimenter.

In the laboratory, in order to ensure the accuracy and safety of the experiment, the experimental operation should be carried out according to the amount of different solutions that should be added in advance or have been calculated in the instructions. After the experiment is done, it should be timely marked on the test tube and other containers with markers to avoid the danger caused by misuse.

Before entering the laboratory, we received systematic and professional skills training. Throughout the entire experimental process, we strictly adhered to laboratory rules and regulations, effectively preventing potential safety hazards. This not only ensured the rigor of the experiments and the safety of the laboratory but also, based on our prior literature review, allowed us to design non-toxic and harmless engineered bacteria, thereby ensuring the safety of both the experimental process and outcomes. Ultimately, we ensured that the products produced are also safe and harmless.

[1]Grozdanov, L., Raasch, C., Schulze, J., Sonnenborn, U., Gottschalk, G., & Hacker, J. (2004). Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917. Journal of Bacteriology, 186(16), 5432-5441.

[2]Gorgieva, S., & Trček, J. (2019). Bacterial cellulose: Production, modification and perspectives in biomedical applications. Nanomaterials, 9(10), 1352.

[3]Helenius, G., Bäckdahl, H., Bodin, A., Nannmark, U., Gatenholm, P., & Risberg, B. (2006). In vivo biocompatibility of bacterial cellulose. Journal of Biomedical Materials Research Part A, 76(2), 431-438.