Team:TU Kaiserslautern/Description

Description

Chlamy Yummy

In the last decades we have been drowning the world in plastic. This has led to an enormous mass of trash polluting our environment. The accumulation affects every organism from bacteria to humans1! It can be found everywhere, from the ground of the Mariana trench to way up in the atmosphere. Even in the arctic the snow contains plastic!

Even though this is already unacceptable to us, we know it’s going to get worse and worse in the upcoming years, as the plastic production is still increasing. The plastic produced now will have an impact on our planet for more than half a millennium. Even though all of it was produced in the last century2!

But a life without plastic is not imaginable. It is a ubiquitous part of our everyday lives. Only because of its stability, light weight and flexibility the development of our society as we know it was possible. Especially in medicine, transport, science and industry the progress we achieved wouldn’t have been feasible without it. Thus, we need a solution for the increasing plastic pollution to not further strain nature.

Fortunately, there are already great projects to eliminate plastic waste from our oceans, for instance the "ocean clean-up project"3. But it is not clear what should happen to the collected plastic. Through saltwater and UV radiation, a massive quality loss occurs, so recycling this waste is not yet an option4. Burning it would be a waste of our planet's resources and would only pollute it further. Therefore we had the vision of an environmentally friendly approach on recycling PET through synthetic biology.

When investigating enzymatic PET degradation, we came across the PETase and MHETase discovered in Ideonella sakaiensis by the japanese Yoshida work group (Yoshida et al., 2016). By secreting the PETase, the bacterium degrades polyethylene terephthalate (PET) into mono-2-hydroxyethyl terephthalate (MHET). Afterwards the MHETase hydrolyses MHET to terephthalic acid (TPA) and ethylene glycol (EG). The bacterium can live off these products as its sole carbon source. This shows the incredible adaptability of nature to a substrate that didn't even exist 50 years ago.

For our project design, we decided to take advantage of the green alga Chlamydomonas reinhardtii (Chlamy) and the modular cloning system (MoClo)5. This system allows for an easy and fast assembly of gene constructs by using standardized fusion sites. This makes the exchange of parts in laboratories all around the world possible. Even though this system is already optimized for Chlamydomonas reinhardtii it is not the only advantage. Its fast growth and the potential for posttranslational modifications allow its use for revolutionizing projects.

By combining the benefits of the expression in our chassis Chlamy and the enzymes PETase and MHETase we created our project Chlamy Yummy - A revolutionizing plastic degradation method.

We genetically modified Chlamydomonas by introducing the genes of both enzymes combined with a secretion signal and the SP20-tag into it. Hoping that increasing the temperature would translate to faster degradation, we implemented Prof. Zimmermann’s idea into our project. Therefore, our transformants were screened to investigate the influence of different temperatures and light intensities on secretion. By doing so we established Chlamydomonas as a secretion chassis. As it shows a spontaneous attachment to PET particles, the green algae make for a perfect organism for producing PET-degrading enzymes. Hence, the PETase and MHETase are directly at the site of action and the highest amount of enzyme is at their appropriate substrate. More importantly, when we created a model about the protein attachment to PET, we were able to demonstrate that the PETase spontaneously binds to PET when brought into immediate proximity. The emerging products TPA and EG are going to be used for the synthesis of new PET, without the quality loss of other recycling methods. Thus, we provide the society with new plastic, that can be utilised as virgin PET. Also Chlamydomonas is perfectly adjusted to grow in a continuous culture, so a constant degradation of PET can be achieved.

Project Inspiration

When we newly formed the iGEM Team of the TU Kaiserslautern we had no idea what kind of project we wanted to work on. We were not really familiar with other iGEM projects, as we had only recently learned about iGEM at all. So we visited the european iGEM meetup 2018 in Munich, where we were inspired by so many ingenious ideas and where we already met our future mentor Rene Inckemann, who further encouraged us to let our ideas flow freely.

Back in Kaiserslautern, we sat down together to brainstorm for ideas. The only thing we knew: With synthetic biology, everything is possible and no idea is too daring. Among the ideas for issues to work on were alzheimers, harmful biofilms, CO2 sensing, PET recycling and digestion of styrofoam. With these loose ideas we started to get more concrete. As most of our team members were environmentalists, PET-recycling was from the start one of the project ideas we were most passionate about. After all, there is hardly any issue more current than the pollution of our environment with plastic. It felt like a stroke of destiny when we stumbled upon the PETNET by iGEM UMaryland 2018, a summary of all successful and unsuccessful projects dealing with PET degradation so far. And after in 2017, the enzymes PETase and MHETase had just been discovered, the PET digestion was a hot topic again. We felt like we were at the right place at the right time.

We talked to our PI Prof. Schroda about our vision and he was immediately on board. His model organism C. reinhardtii was perfectly appropriate as a chassis for this job. It even attaches spontaneously to microplastic, so optimal enzyme delivery could be ensured. The recently developed MoClo part collection meant that C. reinhardtii was already on the starting blocks to become a legitimate new iGEM chassis.

As for our goals, we knew that we could not make the tons and tons of plastic in the oceans disappear at once. Seeing that this was not our jobs as scientists, we set us the goal to provide a proof of principle for our biorecycling. We intended to prove that we could degrade PET, and to quantify and purify the emerging products. Those goals appeared to us as ambitious enough for half a year of time.

We deeply believe that to drive synthetic biology forward, we have to better its acceptance with policy makers and the public. This can be done by presenting possible solutions to problems that so far seem unmanageable. That is partly why we chose PET-degradation as our project, as the issue of the ongoing plastic pollution is highly topical, however there is no solution in sight. We also tried to better synbios reputation in our extensive public engagement work, which we saw as an essential part of our protein.

References

iGem 2019 © Jules