Team:TU Eindhoven/Safety
Laboratory safety
For the iGEM competition, safety is an important aspect. Many of our team members have worked in the laboratory before, so we already knew many safety rules. However, to refresh our memory, we all received safety training. We went to the Biological Safety Officer at our university, Moniek de Liefde-van Beest, who is responsible for the organization of the safety training. We followed a safety training from her and received our Safe Microbiological Techniques certificate. The safety training included general rules for working in the laboratory, emergency procedures and risk classifications. As a part of the safety training, we had to do an assignment where we had to assign risk classifications to imaginary experiments, and we had to explain emergency rules in different situations.
Following the safety training, we performed a risk assessment to determine the risk classes of the organisms we were going to work with. We concluded that we would be working with risk level 1 organisms, which would mean that we would be able to safely work in the ML-I laboratory at our university. We planned on working with E. coli strains (K12 & BL21), which are laboratory strains of risk level 1. These organisms do not pose a threat to humans in the lab or outside the lab. In addition, we planned on working with T7 bacteriophages, which are lytic bacteriophages that target E. coli K12. Bacteriophages do not pose a threat to humans as well. However, in the ML-I laboratory where we would be working, many colleagues perform experiments using E. coli strains, so bacteriophages could infect and destroy the experiments of others. Therefore, we decided to work in a different laboratory at the Queen Astrid Military Hospital in Brussels to protect the experiments of other people.
Before we started working in the laboratory, we received an introduction from Peggy de Graaf-Heuvelmans, who is the lab technician of the BioLab at the TU/e. She informed us about the general rules for working in this specific ML-I lab, responsible persons and emergency contacts and routes. In addition, we received a tour of the laboratory where safety aspects were emphasized.
Furthermore, our PhD supervisor was present in the laboratory when we worked there. She performed new experiments with us to teach us how to perform them safely. She helped us during other experiments as well.
Project safety
It is also important to think about safety concerning the design of our system. We integrated this into our project by participating in the Safe-By-Design assignment of the Dutch National Institute for Public Health and the Environment (RIVM). For this assignment, we spoke with several stakeholders about the design of our detection system. Many of these experts also gave us feedback about the safety of our design, but this was mostly focused on the application of our system in the future. Most experts emphasized that the use of GMOs outside the laboratory should be minimized. In our designed system no GMOs are used outside of the laboratory which is a safety benefit of our system. In addition, it is safer to design a system that uses patient samples instead of a system that needs to be integrated inside the body, which is another safety benefit of our system. However, risks would still be present in handling patient samples. These are aspects that need to be thought of in a later stadium during the development of a device.
Biosafety
Organisms
Different E. coli strains have been used in the project. NovaBlue bacteria (K12 derivative) were used for plasmid amplification and BL21(DE3) cells were used for protein expression. After Gibson Cloning, XL10-Gold cells were used for transformation. The dCas9 plasmid obtained through Addgene was delivered in Mach1 cells (E. coli W derivative, another lab strain generally considered safe).
Vectors
The pET28a vector was used for protein expression. This vector contains a ribosome binding site and a lac promotor to enable inducible protein expression. The pSB1C3 vector was used for the creation of the BioBricks.
Antibiotics
Antibiotics are useful to select the cells that contain the correct plasmid. Kanamycin was used in case of plasmids with pET28a backbone, and chloramphenicol was used for the pSB1C3 vector.
