Team:TU-Eindhoven/Safety/Project

iGEM TU Eindhoven

Project Safety

General safety in the lab is very important, but it is also important to make a design that is inherently safe. The term for this is safe by design. With help of the Dutch National Institute for Public Health and the Environment (RIVM) we formed an opinion about safe by design and how it could be implemented in our project. More information about safe by design and our collaboration with the RIVM can be found here.

The concept of safe by design is to make sure the health and safety risks of the product are addressed from the beginning of the design process. This way, risks can be prevented instead of considered afterwards. Especially in synthetic biology, safe by design is very important. As multiple changes are made, organisms are further deviated from what nature has created. So, we think safe by design should be used in every design or research regarding synthetic biology. To get a better understanding of the term safe by design and how it fits in the current legislation, we did a stakeholder analysis which can be found here.

Our project is also connected to safe by design. We are not developing a bacterial system in which various safety mechanisms can be included, but a whole protein. For proteins, safe by design can be applied in a limited way. We made sure our protein was not toxic, or harmful for the environment. However our scaffold proteins can be used as a tool to implement safe by design in other, bigger researches. See for example our future application scenario T cell therapy in which one of our scaffold proteins can function as a kill switch, thus providing safe by design. There are numerous other cases in which our scaffold can work as a tool for safe by design.

Fusicoccin

Fusicoccin is crucial for our scaffold to work as designed. As described in the background information, fusicoccin is a toxin and it is not proven whether it is safe to use or not. Research showed that fusicoccin in a concentration of 12 times larger than the necessary concentration did not cause any measurable negative effects on the HeLa and HEK293T mammalian cell lines.1 To ensure safe use of fusicoccin in bacterial or mammalian cells, more research on its toxicity is still needed.

Risks of our project

Due to the modularity of the scaffold proteins we designed, they can be used for almost every case in which proteins need to be brought together. On one side these possibilities are a great advantage. On the other hand, when everyone can use these scaffold proteins, they might be used for the wrong purposes. To prevent this from happening, permits around genetic modification should be closely monitored to make ensure they are not misused.

An interesting question is whether you could design something that cannot be misused. With the current technology a lot of things can be used against their real purpose. The goal must be to design things in a way this cannot happen. Whether this is possible or to what extent it is possible to check whether it has been successfully done is still unknown.

Used organisms, vectors and antibiotics

Organisms – Several strains of E. coli have been used in this project. For the amplification of plasmids, the plasmids were transformed in Nova Blue (K-12). For the Gibson Assembly, NEB 5-alpha E. coli is used. For the introduction of mutagenesis, plasmids are transformed into XL10-GOLD. For protein expression, the plasmids are transformed into BL21 (DE3). All of these strains belong to the risk group 1, since they are non-pathogenic strains and will not survive long outside the lab.

Vectors - For our project, we used the pET28a vector, because a lac promotor is built into this plasmid, which makes controlled protein expression possible. For creating the BioBricks of our project we made use of the PSB1C3 vector.

Antibiotics - Different antibiotics are used in order to “kill” bacteria which are not wanted for growing in the cultures. In our case, kanamycin is used so that only the desired bacterial strains with a pET28a plasmid will grow on the agar plates. For the BioBricking part, chloramphenicol is used to ensure that the every bacterial strain contains the pSB1C3 vector.

References
  • [1] Skwarczynska, M., Molzan, M. and Ottmann, C. (2012). Activation of NF- B signalling by fusicoccin-induced dimerization. Proceedings of the National Academy of Sciences, 110(5), pp.E377-E386.

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