Overview
Since we were working with genetically modified organisms (GMOs), safety was a very important topic in our lab. It was necessary to work consciously and safe, to ensure that neither the environment nor humans are harmed. Our genetically modified alga may not get outside of the lab. Because of this, we needed to take precautions to prevent Chlamy from escaping. In case our alga were released into the environment we also made provisions against its survival in the wild.
You can find our completed safety form here.
Lab Safety
To begin, every team member got a safety briefing on working in the wet lab by a qualified supervisor before we started working. We were instructed in general lab safety and how to handle dangerous situations.
Furthermore, we were working in S1 (German biosafety level 1) laboratories. This means there are no risks for the environment and humans1. At S1 laboratories it is important to wear protective clothing like lab coats, a well as safety goggles and gloves when working with toxic substances. Additionally, long hair should be tied back, long pants should be worn, as well as closed shoes. Before entering and leaving the lab, everyone should wash and sterilize their hands2. Our lab has all required safety equipment needed in a S1 laboratory.
General Chlamy Safety
Safety was a key factor in choosing a suitable organism for our project. We were happy to note that Chlamydomonas reinhardtii, a unicellular green alga in risk group 1 and white listed4 for iGEM 2019, was the organism our PI worked with as well, providing us with the ideal organism for our project. C. reinhardtii is a well-studied model organism since the 1940s4 and is considered to be non-toxic for humans5 and the environment.
Project-specific Chlamy Safety
Our whole project is based on the idea of degrading PET into its monomers EG and TPA to purify TPA to produce new PET. Because of that, we decided to put our C. reinhardtii in a bioreactor to work under optimal conditions. A bioreactor is also the best choice to avoid contamination of the environment with our alga because it is a closed system. Besides, the C. reinhardtii strain we used for our experiments is because of the absence of cell wall and flagella not very robust compared to the wild type.
Moreover, the constructs we created have no selection advantages. This means if our alga got outside the laboratory they wouldn’t thrive, so they wouldn’t cause any harm to the existing alga in the environment. Furthermore, there is only a very low horizontal gene transfer of GMO plants known meaning the risks of horizontal gene transfer in Chlamy is insignificant6. Because we wanted to ensure that no alga harmed the environment, we decided to use an auxotrophic C. reinhardtii strain that isn't able to produce arginine by itself. With this strain, we ensured that the alga would die outside of lab conditions.
We also thought about using the Supernova kill switch by the Kazakhstan 2017 iGEM Team. However, we decided that it wasn't necessary because mutations could appear and disable the kill switch. The use of an auxotrophic strain is thus more reliable and safe.
To test the auxotrophicity of the CW325 strain, we made a growth test and a drop test. For the growth test, we measured the cell counts of the CW325 strain cultured with and without arginine, as well as a control UVM4. The test started with a cell count of 2*105 cells per ml. Both the control and the CW325 cultures with arginine grew as expected and reached the plateau phase after 5 days. The CW325 cultures without added arginine didn’t grow because of the lack of arginine.
Growth test of the CW325 arginine strain (in green with arginine and in red without arginine added). UVM4 was used as control.
Besides the growth test, we also made a drop test as orientation for sensitivity of the growth of CW325. In this test we found out which concentration of arginine is needed for the auxotrophic strain to grow. We made plates with different arginine concentrations. These were 0 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml and 100 µg/ml of arginine. The CW325 strain and the UVM4 as control were put on the plates. We chose 1000 cells/µl, 10,000 cells/µl and 100,000 cell/µl as start for the test. As expected, the CW325 on the plate without arginine didn’t grow, while the control did. At any higher tested concentrations the auxotrophic strain grew as quickly as the control.
Shown is the drop test of our arginine auxotrophic Chlamydomonas reinhardtii strain. You can see the cell count per µl on the plates and how much arginine was used in each plate. As expected, the arginine auxotrophic strain does not grow without added arginine
Ethics
Biotechnology and especially genetic engineering are sensitive topics considering ethics. It was very important for us to work responsibly towards the environment and humans.
Because plastic pollution is such an important topic nowadays, it was essential for us to fight this problem with an environmentally friendly method, since most existing methods to degrade plastic are ecologically harmful and not functionally adequate.
Also, in Germany there are strict laws considering genetical engineering (called the Gentechnikgesetz7. Making sure not to introduce GMO’s into the environment is one part of these regulations
Beyond that, we organized a panel discussion together with the ethics committee of our university and invited Prof. Wolfram Henn, a member of the german ethics board as a guest speaker. This was an amazing opportunity to exhange with people of different age groups about ethics in biology.
Literature
- https://www.gesetze-im-internet.de/gentsv/BJNR023400990.html
- https://www.mh-hannover.de/fileadmin/organisation/beauftragte/biologische_sicherheit/webanlagen/s1anforderung.pdf
- https://2019.igem.org/Safety/White_List
- Harris EH. 1989. The Chlamydomonas Sourcebook. San Diego: Academic. 780
- https://journals.sagepub.com/doi/full/10.1177/1091581817746109?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed
- https://www.ebr-journal.org/articles/ebr/abs/2008/03/ebr0742/ebr0742.html
- https://www.gesetze-im-internet.de/gentg/
