Our project uses strains and reagents that are used in biosafety level 1 laboratories. We use non-pathogenic derivatives of E. coli strain (in the event of accidental or otherwise release, the health risks are minimal). Our BioBricks are not supposed to confer pathogenic attributes to E. coli, and there is no data to suggest that they could have any detrimental effects on the environment. At that level, there are no special precautions required, other than those specified for work in laboratory.
Safe Lab Work
Students participating in this project received safety and hygiene training to start the project. The safety training consisted of a presentation covering the various aspects of safety found in molecular biology laboratories. Students were also supervised by instructors throughout the duration of the project. When it is necessary, gloves, glasses and other appropriate personal protective equipment were worn during experiments. The laboratory is equipped in case of fire.
Safe Project Design
Our project also involves regular use of BET, a DNA-intercaling agent known to cause cancer, and the use of UV light for visualization of electrophoresis gel. We wear gloves and a coat to manipulate BET and an appropriate glass for UV. To manipulate our chemistry products, we use nitrile gloves, glasses and coat. We must prepare culture with antibiotics, which could be harmful to humans only in large doses. We also work with Bunsen burner to maintain a sterile environment, which do involve having an open flame on the lab bench.
1. Your new parts
We used four different strains of E. coli: DH5α, MG1655 (clpP::Cm, Z1 and zad220::Tn10).
From the instructions, we understood that only new parts containing new protein coding DNA not already registered in the registry should be included. Our new biobricks consist of composite part assembled from Biobricks already in the registry. Thus we did not include them in the table.
2. What is your chassis organism?
E. coli (lab strains that are not harmful to humans), mainly E. coli DH5α.
3. Do you plan to experiment with any other organisms, besides your chassis?
4. How will your project work?
Most synthetic biology projects involve a risk of GEOs dissemination outside the laboratory, which raises environmental issues. To prevent such a risk, our project aims at designing a biosafety system that would prevent or at least dramatically limit the chances of accidental GEO spreading. The system consists in both a physical confinement which still allows the GEO to carry out its main function, and a temperature-based containment which ensures the survival of the organism in a restricted temperature range (a RNA thermometer and a heat-sensitive repressor control the expression of a TetR repressor located upstream of three essential genes). We chose "Escherichia coli" to implement the temperature-based system since it is widely used in iGEM projects.
5. What risks does your project pose at the laboratory stage? What actions are you taking to reduce those risks?
Risks for the safety and health of team members, or other people working in the lab:
All bacteria (Escherichia coli DH5a and Escherichia coli MG1655) used in our project belong to the risk group 1, so they do not present any risk for the health of the team members. The only real risks come from the manipulation of certain chemicals (such as BET or phenol/chloroform) and the Bunsen burners. Manipulation of agarose gels containing BET were handled with gloves. Phenol/chloroform extraction were carried out under chemical fume hoods, wearing gloves. Regarding gas handling, the lab was equipped with mechanical ventilation and a main on/off valve located outside the lab and turned off every day.
Risk for the environment:
All cultures (solid and liquid), soil, marine and river waters used for the survival study and the small materials used to manipulate living cells (pipette tips, tubes...) were autoclaved before being disposed of. Agarose gels containing BET and phenol/chloroform were disposed in special containers.
6. How would your project be used in the real world?
The initial purpose was to create a device that could be used by the igemers in process to secure their project.
7. What risks might your project pose, if it were fully developed into a real product that real people could use? What future work might you do to reduce those risks?
The physical containment made of silica may break, releasing cells in the environment. The strength of this containment will need to be assessed and it might need to be improved (material strength, shape...). We only worked on one kill-switch system, but we are aware that this is not sufficient to ensure the death of the cells if accidentally released in the environment. Several independent systems will be required, notably to ensure the degradation of the DNA of the dead/dying cells.