Biosafety is a top priority in our iGEM project, focusing on both strict laboratory containment and engineered safeguards to prevent any release of genetically modified organisms (GMOs). In the lab, we follow rigorous biosafety protocols, such as Biosafety Level I (BSL-I), ensuring minimal risk through controlled handling, proper waste disposal, and stringent sterilization practices. To further enhance safety during practical applications outside the lab, we have designed a suicide system for our engineered organisms. This system ensures that, in the event of accidental release, the organisms are unable to survive in external environments, thereby preventing any potential gene leakage into the ecosystem.
A lack of biosafety can lead to disease outbreaks, environmental pollution, and unreliable research results. Proper biosafety awareness helps prevent accidents, ensures operational efficiency, and maintains the accuracy of experimental outcomes.
In our iGEM team, we prioritize biosafety by following standardized procedures, using appropriate lab facilities, and providing comprehensive training on risk prevention and emergency responses. All team members receive comprehensive biosafety training, including risk assessment, safe handling of organisms, and emergency response protocols. Additionally, our team conducts thorough risk assessments of all biological materials used in our projects to ensure compliance with biosafety protocols and prevent unintended release into the environment. Even as students, fostering responsibility for biosafety is key to our current and future research endeavors.
Before starting our experiments, we selected Escherichia coli Nissle 1917 as our foundational microorganism due to its well-documented safety profile and long history of use in clinical and research settings. This strain has been extensively studied for over a century since it was identified in 1917 for its strong antagonistic activity in the gut.
Nissle 1917 is a non-pathogenic probiotic strain with well-documented clinical applications. It has been safely used to treat various intestinal disorders such as ulcerative colitis and diarrhea, and unlike harmful E. coli strains, it does not carry toxic or antibiotic resistance genes. Furthermore, it enhances gut barrier function, inhibits the growth of harmful bacteria, and promotes the balance of healthy gut flora.
Given its fully sequenced genome, E. coli Nissle 1917 is well-suited for genetic manipulation, allowing us to introduce specific modifications in a controlled manner. These manipulations are carefully designed and reviewed to avoid introducing any new risks, such as antibiotic resistance genes or virulence factors, thereby ensuring the strain remains safe for lab use.
Throughout our project, we follow strict biosafety procedures to handle E. coli Nissle 1917 safely in the lab, ensuring that no engineered strain is released into the environment or poses any risk to public health. This attention to biosafety allows us to leverage the benefits of Nissle 1917 in synthetic biology applications confidently and responsibly.
Human epidermal growth factor (hEGF) is a small polypeptide hormone composed of 53 amino acids with a molecular weight of approximately 6,000 Daltons, containing three disulfide bonds that influence its bioactivity. It plays a key role in cell proliferation, differentiation, and migration, particularly in the growth and repair of epithelial cells. hEGF is widely used in the medical and cosmetic industries, such as promoting the healing of burns and wounds, treating gastric ulcers and corneal injuries, and improving skin texture and repairing skin in cosmetic products. While hEGF is considered an important growth factor, studies have shown that high concentrations may lead to potential toxicity, so dosage control is essential. The dosage of hEGF has been carefully considered during the project design process to avoid potential toxicity.
mazF is a toxin protein produced by bacteria (such as *E. coli *), and it is part of the mazEF toxin-antitoxin system. This system consists of two genes: mazE (encoding the antitoxin) and mazF (encoding the toxin). mazF is an mRNA endonuclease that cleaves mRNA at specific sites, thereby inhibiting protein synthesis and ultimately leading to cell death.
The rhamnose promoter (pRHa) is an inducible promoter commonly used for regulating gene expression. Its activity is controlled by the concentration of rhamnose (L-rhamnose), a naturally occurring six-carbon sugar. In synthetic biology and molecular biology experiments, the rhamnose promoter is often used to precisely control the expression of exogenous genes.
Our designed suicide system relies on the dynamic regulation of the toxin-antitoxin system. As shown in Figure 2, the expression of mazF (encoding the toxin) is controlled by a promoter. When this promoter is activated, mazF expression produces the toxin mazF, an mRNA endonuclease that cleaves cellular mRNA, blocking protein synthesis, and ultimately leading to cell death. However, to prevent premature cell death when the suicide mechanism is not needed, we incorporated mazE (encoding the antitoxin) into the genetic circuit, whose expression is controlled by the rhamnose-inducible promoter (Prha).
mazE neutralizes the toxic effects of mazF, protecting the cell from damage. When rhamnose is present, the Prha promoter is activated, mazE is expressed, and the antitoxin mazE binds to mazF, inhibiting its toxic effects, ensuring cell survival. As rhamnose is absorbed by the human gut, leading to a lack of rhamnose, mazE expression stops, the mazE antitoxin gradually degrades, and the mazF toxin regains its activity, initiating mRNA cleavage and triggering cell death.
This system allows the survival or death of E. coli to be controlled by external regulation (presence or absence of rhamnose), enabling safe management and control. Additionally, if the bacteria are excreted via feces into the external environment, where rhamnose is absent, mazE expression will also cease, leading to bacterial death due to the lack of mazE antitoxin. This provides an extra layer of safety, ensuring that the bacteria do not survive outside the host environment.
Before entering the laboratory, we organized and participated in a comprehensive and systematic laboratory safety training. The training covered topics such as proper handling of chemicals, biological safety measures, emergency response procedures, and the correct use of laboratory equipment. Through this training, we not only became familiar with various potential risks in the laboratory but also acquired skills to handle emergencies, ensuring that we can effectively prevent accidents and safeguard both the team members and the smooth progress of experiments in the future.
During experimental operations, all team members work under the supervision of experienced laboratory instructors to ensure safety throughout the process. When entering the lab, we always wear lab coats and rubber gloves, strictly following the protective requirements (Figure 2). Additionally, we avoid bringing food and drinks into the lab to maintain a clean and safe environment.
When using the biosafety cabinet, we turn on the UV light 30 minutes before the operation to sterilize the workspace. Before starting, we also use 75% alcohol to thoroughly disinfect our hands and the items to be placed inside the cabinet, ensuring a sterile environment. Each experimental sample is clearly labeled and stored at the appropriate temperature to prevent contamination or degradation.
As for general waste, we dispose of it in designated laboratory waste bins, and a dedicated staff member regularly collects and handles these materials, ensuring that waste disposal complies with laboratory safety standards, thus maintaining cleanliness and environmental sustainability in the lab.
Figure 2 shows a fire extinguisher and fire alarm device that we captured. At the top near the wall is a fire alarm button, which can be manually activated to trigger the fire alarm system in case of an emergency. Below it is a fire extinguisher box containing a fire extinguisher, which can be used to put out fires during the early stages. Both devices are common laboratory fire safety equipment, designed to prevent the spread of fire and provide quick response measures.
Figure 3 shows an emergency shower and eyewash station, which is very common in laboratories. In the event of chemicals splashing onto the eyes or skin, this device can be used for rapid rinsing to minimize harm. At the top is an emergency shower for full-body washing, and below is an eyewash station specifically designed to rinse the eyes, ensuring that chemicals are quickly removed in case of an emergency.
This project strictly follows biosafety regulations, selecting the safe chassis microorganism E. coli Nissle 1917, and applying dosage control to human epidermal growth factor (hEGF) to ensure safety. The designed mazE/F toxin-antitoxin suicide system is regulated by the rhamnose promoter, ensuring controllability of the strain. Before entering the laboratory, we conducted comprehensive safety training covering chemical handling, biosafety measures, and equipment use. Additionally, the laboratory is equipped with fire extinguishers, fire alarm devices, and emergency shower and eyewash stations, ensuring safety protection in emergencies and supporting the smooth progress of the experiments.