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Lab safety

There are four BioSafety levels, ranging from minimal risk to humans (level 1) to causing severe to fatal diseases (level 4)^[1]. Our lab is classified as a BioSafety Level 1. Most of the work done at the lab involves the recombinant expression of proteins in Escherichia coli (E. coli) or yeast expression systems and the application of bacteriophages for phage display. Furthermore, cloning and plasmid expansion is also part of the work done at this lab.

When working with GMOs, it is important to work clean, carefully, hygienically and know safe microbiological techniques (SMT). Before starting an experiment, the risks should also be analyzed. Next to that, it is also necessary to check if the experiments are included into the license, this can be done with the PI or the biosafety officer. A biosafety officer is the main responsible for biosafety. At our department the biosafety officer is Moniek de Liefde-van Beest.The PI of our department is Maarten Merkx.

The SMT ground rules are:

Always work according to the rules

Keep doors and windows closed

Wear a lab coat

Gather everything and place it orderly

Avoid hand-body (e.g. face) contact

Do not eat or drink in the laboratory

Avoid aerosol formation

Do not pipette orally

Disinfect non-disposable materials, dispose other materials correctly

Clean up your workspace and desinfect the surface

Wash your hands when leaving the lab

The more elaborate rules can be found here.

Project Safety

Besides working safe in the lab, it is also important to think about the applications and risks of your project beforehand. This is called safe-by-design and together with the Dutch National Institute for Public Health and the Environment (RIVM) we looked into this and thought about ways to incorporate this into our project. More information about Safe by Design can be found here.

For our project we use E. coli as a host organism, which is an organism of risk group 1. This organism is then transformed to express our proteins constructs: a scaffold protein (14-3-3) and its binding partner (CT52). Since E. coli is only used to produce the wanted proteins, we are not developing a bacterial system in which safety mechanisms like a kill switch can be induced. However, we did look at the safety of our proteins and at the safety of the network that is formed, more information can be found in the background and in Safe by Design. Furthermore, we also looked at eventual applications and what kind of problems may arise. For our system, we envision it as a gel that can encapsulate tumor cells. To make sure that this gel forms locally, a specific cleavage site can be incorporated in one of the protein constructs. More information on the application scenario can be found here.

Fusicoccin

Fusicoccin is a small molecule produced by the fungus Fusicoccum amygdali. In our project, it is used as a stabilizer for the interaction between the scaffolding construct and the binding construct. Research has shown that fusicoccin in a concentration 12 times larger than the minimum concentration did not cause measurable negative effects on HeLa and HEK293T mammalian cell lines.^[2] However, more research on its toxicity is needed to ensure safe use of fusicoccin in bacterial or mammalian cells.

Risks of our project

Since our project does not involve developing a bacterial system, no hazardous risks are present. However, the use of fusicoccin as a stabilizer is a risk in the eventual application of our project. Bacteria naturally lack the 14-3-3 proteins, so fusicoccin would not cause a risk, while in humans fusicoccin could interfere with natural processes. Therefore, fusicoccin would not be safe to use in the human body. Other inducers, such as proteases, can be incorporated in the design of the protein constructs instead of fusicoccin.

Biosafety

Organisms

Different strains of E.Coli have been used in this project. For plasmid amplification, the plasmids were transformed into Nova Blue (K-12). When performing Gibson Assembly, NEB 5-alpha E.Coli was used. For protein expression, the plasmids were transformed into BL21 (DE3). All strains are attenuated E.Coli strains and can be handled at Biosafety Level 1.

Vectors

The pET28a vector was used in this project. A lac promotor is built into this plasmid, making controlled protein expression possible. The pSB1C3 vector was used for creating the BioBricks.

Antibiotics

The use of anti-microbial resistance factors are a useful tool to quickly determine which cells received the correct plasmid. In our project, kanamycin was used to check for the presence of the pET28a vector. For BioBricking, chloramphenicol was used as a resistance factor to check for the presence of the pSB1C3 vector.

^[1]Laboratory Biosafety Manual, World Health Organization, Third Edition.
^[2]M. Skwarczynska, M. Molzan and C. Ottmann, "Activation of NF-B signalling by fusicoccin-induced dimerization", Proceedings of the National Academy of Sciences, vol. 110, no. 5, pp.E377-E386, 2012.

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Team:TU-Eindhoven/Safety