Biosafety is an important part of project design. In the case of Muscure, a clinical application project, we primarily focus on project safety design from four aspects: biological design, production, transportation, and use.
We have also consulted relevant professionals regarding project safety. To learn more, please refer to Human practice.
In project design, we double our biosecurity through nutritional deficiencies and suicide genes. We used four deficiencies of yeast with four nutritional deficiencies as the engineered bacteria. The yeast chassis we use has defects in lysine, leucine, uracil and histidine synthesis. After transforming plasmids, our engineered yeast contains defects in histidine and uracil synthesis. As a result, engineered yeasts can only survive under laboratory conditions at present, effectively preventing the leakage of engineered bacteria into the environment.
Fig 1 Nutritional deficienciy
We also designed a suicide system using bile acids, the gut marker molecules. Bile acids can bind to and activate FXR proteins, and activated FXRs can act as transcription factors to activate CI protein expression downstream of the BSEP promoter. CI protein can inhibit the expression of the suicide gene MazF downstream of the PL promoter, thereby inhibiting cell suicide. When the yeast is not in the intestinal environment (no bile acid signaling), the successful expression of MazF leads to cell death, achieving the purpose of controlling the yeast in the gut.
Fig 2 The design of suicide system
According to our design, therapeutic engineered yeast will be produced in a large-scale specialized microbiological facility. Specialized facilities must have the appropriate equipment and trained personnel. What's more, the laboratory and premises during the production process must adhere to international biosafety standards and ensure strict biosafety levels, such as BSL-2 or BSL-3. The cultivation of Saccharomyces cerevisiae must take place in controlled bioreactors to minimize the risk of leakage. Personnel must undergo specialized training and strictly follow operating procedures, including wearing protective gear and using biosafety cabinets. Waste must be handled and disposed of in accordance with the standards for biohazardous waste to prevent any leakage of biological materials.
We consider the transportation process to be one of the most crucial aspects of biosafety in bacterial therapy projects. After thorough investigation and research, we have determined that using lyophilized yeast powder for transportation is the optimal choice. Lyophilized yeast powder has the advantage of being transported at room temperature without loss of activity. Moreover, such yeasts are less prone to leakage and contamination.
During transportation, double-sealed containers should be employed to minimize the risk of bacterial leakage. Continuous monitoring must be carried out throughout the transportation process to ensure that no leakage or contamination occurs.
For patients, they must use this strain under the guidance of a medical professional or pharmacist and inhale muscone as prescribed. Patients undergoing the therapy must undergo regular monitoring to detect any potential adverse reactions or leaks.
For hospitals, the handling of engineered yeasts is also crucial. Healthcare professionals, including doctors and nurses, should receive specialized training to understand the proper use of engineered yeasts. Hospitals should also procure specialized equipment for storing or resuscitating these strains. Additionally, medical waste such as syringes used during treatment should be separately disinfected to prevent the leakage of engineered yeasts into the environment.
Laboratory safety is an essential component of the iGEM project. We have implemented various measures to ensure the biosafety of the iGEM laboratory for every participant involved.
All wet experiments are conducted at the Tsinghua University Life Science Innovation Laboratory. Following the university's regulations, all experimental procedures adhere to the "Tsinghua University Laboratory Biosafety Management Measures".
Fig 3 Laboratory safety education and examination system
All team members have undergone and passed the laboratory safety education and examination system, qualifying them for participation in wet experiments.
Fig 4 Laboratory safety education and examination system
Prior to commencing wet experiments, instructors and consultants provided detailed instructions on the proper use of laboratory equipment, emphasizing precautions when using hazardous equipment such as high-pressure sterilizers. This safety education has provided us with comprehensive understanding of the safety regulations to be followed in the laboratory and the safety considerations to be taken during experimental procedures.
Fig 5 The slides of safety education
Before participating in wet experiments, all team members have signed safety protocol agreements to demonstrate their understanding of laboratory safety protocols and their commitment to responsible behavior.
To constantly remind everyone of their safety during the experiment, safety signs are posted throughout the laboratory. Specific instructions for the use of the equipment are indicated on the relevant experimental instruments, such as a waste tank for discarding special liquids.
Fig 6 Laboratory equipments
We have implemented a "last person" system, in which the last person to leave the laboratory is responsible for checking whether all experimental instruments are organized or closed and must sign a confirmation form. This ensures our commitment to environmental protection and safety on a daily basis.
Fig 7 "Last person" form
Regular team meetings also involve discussions and summaries of encountered experimental safety and protocol-related issues, such as proper procedures for waste segregation. This continuous practice deepens our understanding of laboratory safety regulations.