Short Introduction:
The very first question every iGEM-Team needs to answer is what their project should be about. Our goal was not only to participate successfully in the iGEM competition but to give something back to both the synthetic biology community and our local community. We regularly encounter plastic pollution in our town and the neighboring forest. Larger scale plastic pollution occurs in countries all over the world. This inspired us to focus on the degradation of plastic by implementation of synthetic biology. Our approach does not suggest that plastic pollution should continue, but it offers an environmentally friendly way to fight the existing plastic pollution. We developed a method to degrade PET cheaply, eco-friendly and infinitely often.
Our project idea focuses on one of the most common plastics, polyethylene terephthalate (PET). With the help of two enzymes we can degrade PET into its monomers - ethylene glycol and terephthalic acid. Since PET is degraded, other components such as plasticizers are released and therefore separated from the degradation products. Another point is that our system is applicable to microplastics, which is a novelty in plastic recycling.
Why use Chlamydomonas reinhardtii?
During our investigation on project design, we found a paper from a japanese work group that described two newly discovered enzymes, PETase and MHETase.1 The enzymes were discovered in the bacterium Ideonella sakaiensis and can degrade PET into its monomers. Those enzymes were perfect for our project, but further research showed that there have been a couple of previous iGEM teams working with these enzymes. But those teams seemed to struggle with their choice of chassis. We were thinking about how we could improve this approach and learn from the previous teams. That's how the idea arose to work with Chlamydomonas reinhardtii. This made us the first iGEM team working with a eukaryotic organism in combination with these two enzymes. Since posttranslational modifications (PTMs) occur in eukaryotic organisms, we expected an improvement of secretion and higher stability of the proteins. Besides, C. reinhardtii already has a secretion system and is a well-established model organism in our work group. It is often used to produce recombinant proteins and is easy to handle and cultivate. Furthermore, C. reinhardtii does not build biofilms that can prevent secretion, which was one of the problems other teams had to deal with. But the best part about using C.reinhardtii as our chassis is that it attaches naturally to microparticles of PET. This increases the concentration of the proteins at their target location and therefore the efficiency of degradation.
We decided to first work with the UVM4 strain which has no cell wall and is therefore well suited for secretion. Later, we switched to the clip strain with a cell wall for experiments in the bioreactor. The clip strain is more resistant against turbulences that occur in the bioreactor.
Chlamydomonas reinhardtii shows spontaneous attachment to PET. Wild-type strain CC-4533 was incubated for 7 days in TAP medium with 1 g PET/l. The cells spontaneously attached to the microplastic surface. This renders Chlamydomonas reinhardtii as a perfect model organism for the production of plastic-degrading enzymes
Modular Cloning System (MoClo): our Working Tool:
The MoClo system is based on the golden gate cloning system and works with type IIs restriction enzymes. These enzymes cut DNA outside their recognition side. This allows for the design of defined overhangs. During the ligation process the recognition side gets excised which results in a scarless construct. The MoClo system is a standardized system for C. reinhardtii, so the exchange between different labs is very easy.
The modular cloning system. MoClo is a standardized synthetic biology tool based on Golden Gate Cloning. It allows the rapid assembly of genetic parts in a predefined arrangement.
The MoClo system is established in our group, therefore we have access to a lot of expertise and support. The greatest advantages of the MoClo system are the possibility to design different parts in a timesaving way as well as being able to switch gene parts easily. Only with the help of this fast ligation system, we were able to get the part collection we worked with. One thing that also must be mentioned is the efficiency of the MoClo system. It allows a fast and highly efficient working process which results in a faster workflow in comparison to standard cloning methods.
Toxicity Test
Before we got started with designing our constructs, we needed to ensure that the degradation products of PET are non-toxic to C. reinhardtii. This was essential to the project. Therefore we tested the two products terephthalic acid and ethylene glycol in different concentrations for realistic results in a physiological range. The toxicity tests were made with three technical and three biological replicates and the amount of cells was measured by using a cell counter device. This ensured us to get more reliable results than measuring with optical density. The cultures were grown under standard conditions for optimal growth. To get the second confirmation of our results we performed a spot test. The toxicity tests were important to check if the project had the potential for later use on an industrial scale.
Our Parts
During the competition, we built 27 level 0, 25 level 1 and 35 level 2 gene constructs. For our enzymes PETase and MHETase, we used the PAR promotor in combination with the RPL23 terminator and for the antibiotic resistance cassettes, we used the PSAD promotor in combination with the PSAD terminator to avoid repetition. The PAR promotor is a fusion promoter and very efficient for expressing proteins in C.reinhardtii.2 To select the transformants, we used the spectinomycin resistance cassette aadA. As a second resistance, we ligated the hygromycin cassette for a co-transformation. For every part, we optimized the codon usage and added introns so that the sequence is suitable for C. reinhardtii. Combining the content of two different publications, we introduced three point mutations: R280A3, S238F4 & W159H4 into the PETase gene that, as we suspected, increase the activity of the enzyme. To detect the proteins, we used the HA-tag. Later, we also introduced the His-tag to our constructs for a better distinction of the proteins as well as purification purposes. We started out with a level 2 construct without secretion signals.
part number Bba_K3002200. Part number Bba_K3002200 harboring the aadA selection marker, the coding sequence of HA-tagged MUT- PETase and the coding sequence of HA-tagged MHETase.
We first tested the cytosolic expression of the proteins. Then we expanded the constructs by the secretion signal cCA.
part number Bba_K3002202. Part number Bba_K3002202 harboring the aadA selection marker, the coding sequence of HA-tagged MUT- PETase equipped with the secretion signal ccA and the coding sequence of HA-tagged MHETase equipped with the secretion signal cCA.
The results showed that the MHETase is secreted, but for the PETase no secretion was detectable. Therefore, we tested different secretion signals in combination with the PETase. We used the secretion signal for the gamete lytic enzyme (GLE) as well as the secretion signal for arylsulfatase, ARS.
part number Bba_K3002210 Part number Bba_K3002210 harboring the aadA selection marker, the coding sequence of HA-tagged MUT- PETase equipped with the secretion signal ARS and the coding sequence of HA-tagged MHETase equipped with the secretion signal cCA.
part number Bba_K3002211. Part number Bba_K3002211 harboring the aadA selection marker, the coding sequence of HA-tagged MUT- PETase equipped with the secretion signal ARS and the coding sequence of HA-tagged MHETase equipped with the secretion signal cCA.
Another approach was to design new constructs of the PETase in combination with the three secretion signals, but without the MHETase.
part number Bba_K3002209 Part number Bba_K3002209 harboring the aadA selection marker and the coding sequence of HA-tagged MUT- PETase equipped with the secretion signal ARS.
part number Bba_K3002207. Part number Bba_K3002207 harboring the aadA selection marker and the coding sequence of HA-tagged MUT- PETase equipped with the secretion signal GLE.
part number Bba_K3002208. Part number Bba_K3002208 harboring the aadA selection marker and the coding sequence of HA-tagged MUT- PETase equipped with the secretion signal ARS.
We analysed the different constructs on their efficiency of PETase secretion. At the same time, we performed fluorescence microscopy to get an idea where the PETase got stuck. Analysis by fluorescence microscopy showed that PETase is stuck in the ER. Further research brought a paper to light that introduced the SP20 tag which increased the yield of a secreted protein from C.reinhardtii.5 We integrated the SP20 tag in our level 2 construct expecting that it might enhance the secretion of the PETase.
part number Bba_K3002212. Part number Bba_K3002212 harboring the aadA selection marker, the coding sequence of SP20-HA-tagged MUT- PETase equipped with the secretion signal ccA and the coding sequence of SP20-HA-tagged MHETase equipped with the secretion signal cCA.
part number Bba_K3002213. Part number Bba_K3002213 harboring the aadA selection marker, the coding sequence of SP20-HA-tagged MUT- PETase equipped with the secretion signal ARS and the coding sequence of SP20-HA-tagged MHETase equipped with the secretion signal cCA.
part number Bba_K3002214. Part number Bba_K3002214 harboring the aadA selection marker, the coding sequence of SP20-HA-tagged MUT- PETase equipped with the secretion signal GLE and the coding sequence of SP20-HA-tagged MHETase equipped with the secretion signal cCA.
To allow the purification of the proteins we combined the SP20 tag with a 8xHis tag.
part number Bba_K3002221. Part number Bba_K3002221 harboring the aadA selection marker and the coding sequence of SP20-His-tagged MUT- PETase equipped with the secretion signal ccA.
part number Bba_K3002222. Part number Bba_K3002222 harboring the aadA selection marker and the coding sequence of SP20-His-tagged MUT- PETase equipped with the secretion signal ARS.
part number Bba_K3002223. Part number Bba_K3002223 harboring the aadA selection marker and the coding sequence of SP20-His-tagged MUT- PETase equipped with the secretion signal GLE.
part number Bba_K3002224. Part number Bba_K3002224 harboring the aadA selection marker, the coding sequence of SP20-His-tagged MUT- PETase equipped with the secretion signal ccA and the coding sequence of SP20-HA-tagged MHETase equipped with the secretion signal cCA.
part number Bba_K3002225 Part number Bba_K3002225 harboring the aadA selection marker, the coding sequence of SP20-His-tagged MUT- PETase equipped with the secretion signal GLE and the coding sequence of SP20-HA-tagged MHETase equipped with the secretion signal cCA.
part number Bba_K3002226 Part number Bba_K3002226 harboring the aadA selection marker, the coding sequence of SP20-His-tagged MUT- PETase equipped with the secretion signal ARS and the coding sequence of SP20-HA-tagged MHETase equipped with the secretion signal cCA.
Analysis of the constructs showed that the secretion of the PETase was enhanced due to the use of the sp20 tag. To reach an equal secretion of both enzymes we designed a fusion protein that can be tested in the future.
The Development of our Screening Process
Since the method of analysing proteins in culture medium wasn’t established in our workgroup, we decided to create a protocol with the help of our advisors. In the following screening process, we improved the protocol to optimize the results. We first started with a TCA precipitation of the supernatant with a volume of 15 ml. Because of the high volumes, the time-consuming process as well as the limitation by the number of reaction tubes fitting into the centrifuge, we switched our protocol. The lyophilisation of the supernatant combined with an acetone precipitation was a faster and easier method. Due to the method we were also able to process smaller volumes of supernatant (2 ml) and more samples at once. The precipitated protein was then resuspended in loading buffer and ready to load onto a SDS-gel. As a last improvement step, we decided to load less of the sample onto the gel which made it easier to compare the signals to each other.
Another important part of the process was to find the best time to let the cultures grow for secretion. We started out with seven days, but after taking samples every day, we figured out that the perfect time for harvesting was four days. After the glycosylation by the SP20 tag, we observed a mass shift compared to signals in samples without the sp20 tag. To assign the observed signals to our enzymes analysis by LC-MS was done.
Analytics
After the successful secretion of the enzymes, we had to test the activity of those. In the beginning of those experiments we used shredded PET bottles as a substrate for the PETase, This method was not successful due to the solid crystal structure of PET bottles. Therefore, we switched to other plastic forms available in the supermarket which are more flexible. After a few weeks, we heard of the working group of Professor Zimmermann in Leipzig. His workgroup uses a special PET film with a higher amorphous protion. In the first approaches, we incubated the cultures of C. reinhardtii with the PET film. The PET particles were weighed and microscoped before and after the incubation. Since the results weren’t meaningful, we changed our method. After the incubation time, we extracted the supernatant with ethyl acetate to concentrate TPA for analysis by reverse HPLC. To get a higher significance of the results, we decided to use the concentrated supernatant and incubated it with the different substrates (PET, BHET, MHET). The samples were incubated for 96 hours at 25°C and samples were taken every 24 hours to test activity in dependence of time. Another side experiment included the expression of enzymes in E.Coli. This was done to find out if the activity of the protein was affected by the glycosylation of a eukaryotic chassis compared to the proteins from E.Coli.
Integrated Human Practice
To learn from other scientists, we reached out to Professor Zimmermann from the university of Leipzig. He has been working with the enzyme cutinase on the degradation of PET. His results showed that the degradation of PET works best at high temperatures. Therefore, we tested the activity of our alga at 25°C and 33°C and our enzymes at 25°C, 30°C, 33°C and 40°C.
Our Aim: the Bioreactor
We wanted to create an environmentally friendly recycling system. To realize this, we established our concept in a bioreactor. This allows us to gather the degradation products for the resynthesis of PET. It also offers the perfect growth condition for our green alga and thereby is cheap and eco-friendly. The concept of a green alga in a bioreactor can be increased to bigger volumes, which could lead to an industrial application. But most importantly, a bioreactor is a closed system. An important point in our safety concept is that there is no contact between our genetically engineered organism and the environment.
References
1Yoshida, Shosuke; Hiraga, Kazumi; Takehana, Toshihiko; Taniguchi, Ikuo; Yamaji, Hironao; Maeda, Yasuhito et al. (2016): A bacterium that degrades and assimilates poly(ethylene terephthalate). In: Science (New York, N.Y.) 351 (6278), S. 1196–1199. DOI: 10.1126/science.aad6359.
2Schroda, Michael; Blöcker, Dagmar; Beck, Christoph F. (2000): The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. In: The Plant Journal 21 (2), S. 121–131.
3Joo, Seongjoon; Cho, In Jin; Seo, Hogyun; Son, Hyeoncheol Francis; Sagong, Hye-Young; Shin, Tae Joo et al. (2018): Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. In: Nature communications 9 (1), S. 382. DOI: 10.1038/s41467-018-02881-1.
4Austin, Harry P.; Allen, Mark D.; Donohoe, Bryon S.; Rorrer, Nicholas A.; Kearns, Fiona L.; Silveira, Rodrigo L. et al. (2018): Characterization and engineering of a plastic-degrading aromatic polyesterase. In: Proceedings of the National Academy of Sciences of the United States of America 115 (19), E4350-E4357. DOI: 10.1073/pnas.1718804115.
5Ramos-Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko (2017): High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii. In: Plant biotechnology journal 15 (9), S. 1214–1224. DOI: 10.1111/pbi.12710.
