Team:Kyoto/Description

Description
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Environmental Pollution by Plastics
We use various kinds of products made of plastics in our daily lives because of its many advantages. To be specific, it is remarkably durable for its weight. However, the trouble is that it is barely degraded in the natural environment, which leads to their unstopping accumulation [1]. Such environmental plastics often exist mainly as tiny fragments and ones that are less than 5 mm are especially called microplastics. As you may know, this is one of the most severe environmental problems that the world is facing now.
Why is This Problem So Serious?
More than 5 trillion plastic fragments, weighing over 250,000 tons were estimated to be floating on the oceans [2]. These microplastics can be mistakenly eaten by marine organisms including endangered species [3]. The surface of plastic is hydrophobic and smooth, so hazardous substances can easily attach to it. When this kind of microplastic is mistakenly eaten by marine organisms, it will do harm to them and this is endangering the ecosystem as a whole [4].
So Where on Earth Do They Come From?
You may know that plastic-made products such as plastic bottles, plastic bags, plastic straws, etc that are thrown away in nature become microplastics. However, most of microplastics come from a remarkably common everyday activity that is hardly recognized: laundry. A research conducted by Imogen E. Napper shows that more than 700,000 microfibers come off from clothes in just one laundry [5]. Moreover, considerable numbers of microfibers released from washing machines do not seem to be captured in wastewater treatment facilities. According to Sherri A. Mason, billions of microplastic particles are released into the natural environment from wastewater treatment plants in the U.S. every day and 60 percent of them are microfibers [6].
Although there has not been an effective way to remove microplastics from the environment, an enormous amount of microplastics still flow into the natural environment. This major issue is what we are tackling in our project.
References
1 Barnes, D.K.A., Galgani, F., Thompson, R.C., and Barlaz, M. (2009).
Accumulation and fragmentation of plastic debris in global environments.
Philos. Trans. R. Soc. B Biol. Sci. 364, 1985–1998.
2 Eriksen, M., Lebreton, L.C.M., Carson, H.S., Thiel, M., Moore, C.J., Borerro, J.C., Galgani, F., Ryan, P.G., and Reisser, J. (2014).
Plastic Pollution in the World’s Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea.
PLoS One 9, 1–15.
4 Rochman, C.M., Hoh, E., Kurobe, T., and Teh, S.J. (2013).
Ingested plastic transfers hazardous chemicals to fish and induces hepatic stress.
Sci. Rep. 3, 1–7.
5 Napper, I.E., and Thompson, R.C. (2016).
Release of synthetic microplastic plastic fibres from domestic washing machines: Effects of fabric type and washing conditions.
Mar. Pollut. Bull. 112, 39–45.
6 Mason, S.A., Garneau, D., Sutton, R., Chu, Y., Ehmann, K., Barnes, J., Fink, P., Papazissimos, D., and Rogers, D.L. (2016).
Microplastic pollution is widely detected in US municipal wastewater treatment plant effluent.
Environ. Pollut. 218, 1045–1054.
How We Decided the Project Theme
As we were having a really hard time deciding the project theme, our team PI advised us to identify a clear goal to help solve a relevant social problem. Then, one of our team members proposed the problem of microplastics. He has somehow wanted to help prevent the unstopping release and accumulation of microplastics ever since he saw a TV program about their potential threats to humans and other animals. Although we did not have a specific idea of how we could deal with it at first, his enthusiasm led us to think about this problem seriously.
So How Can We Help Solve This Problem?
At first, we were thinking about how we could retrieve microplastics from the huge bodies of water such as the world's oceans. We found a website by The Ocean Cleanup that said they have been creating a large-scale floating device to collect plastic wastes floating on the oceans [1]. It says this device is able to retrieve wide-ranging sizes of plastics so much at a time with little by-catch. So we thought we needed to find a different approach using a synthetic-biological system since the world has already started to develop such a highly-efficient large-scale solution.
We also thought of degrading them by using PET-digesting enzyme, PETase, which is found in Ideonella sakaiensis [2]. However, they have a problem with the degradation efficiency. This bacteria were reported to degrade the PET film surface at a rate of 0.13 mg/cm^2/day at 30°C (Fig.1)[2]. Since the cause of its low activity was identified, a lot of research has been done to improve its activity [3][4][5]. There are also many previous iGEM projects that dealt with this topic (See the list).However, since 4.8 to 12.7 million metric tons of plastics were estimated to have entered the oceans in 2010 alone, we even began to think that no matter how much researchers successfully improved PETase's activity, the world has so many plastics to degrade that the idea of degrading itself seemed so unrealistic for the solution [6]. Furthermore, if its activity were not enough, it might just end up degrading them into much smaller “nanoplastics”, which would even make the problem worse (Fig.2).
Fig.1 I.sakaiensis degrades PET film slowly
Yoshida et al. found that I.sakaiensis isolated from PET-contaminated environmental samples degraded PET film at a rate of 0.13mg/cm^2/day at 30°C in the MLE medium.
Fig.2 Microplastics degraded into “nanoplastic”
If plastic-digesting enzyme is not active enough, it might do more harm than good by making much smaller plastic fragments.
Taking these two points into account, we turned to the idea of capturing them before they flow into the natural environment.
Meanwhile, one of our team members came across an interesting paper that a single laundry process was responsible for the production of more than 700,000 microfibers [7]. We also learned from papers that there are several kinds of proteins that are shown to have the plastic-binding property [8][9][10]. So we decided to try to figure out a novel way to remove microfibers from laundry wastewater before they enter the oceans, with the application of these synthetic-biological tools (Fig.3).
Fig.3 Stopping microfibers before being released into the oceans
It requires a huge and well-organized system to retrieve microplastics after they enter the oceans, so we aimed to catch them beforehand.
So this is how we started to work on this strategy!
References
1 The Ocean Cleanup
https://theoceancleanup.com
2 Yoshida S., Hiraga K., Takehara T., Taniguchi I., Yamaji H., Maeda Y., Toyohara K., Miyamoto K., Kimura Y., and Oda K. (2016).
A bacterium that degrades and assimilates poly(ethylene terephthalate).
Science 351, 1196-1199.
3 Furukawa, M., Kawakami, N., Oda, K., and Miyamoto, K. (2018).
Acceleration of Enzymatic Degradation of Poly(ethylene terephthalate) by Surface Coating with Anionic Surfactants.
ChemSusChem 11, 4018–4025.
4 Son, H.F., Cho, I.J., Joo, S., Seo, H., Sagong, H.Y., Choi, S.Y., Lee, S.Y., and Kim, K.J. (2019).
Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation.
ACS Catal. 9, 3519–3526.
5 Ma, Y., Yao, M., Li, B., Ding, M., He, B., Chen, S., Zhou, X., and Yuan, Y. (2018).
Enhanced Poly(ethylene terephthalate) Hydrolase Activity by Protein Engineering.
Engineering 4, 888–893.
6 Jambeck, J., Geyer, R., Wilcox, C., Siegler, T.R., Perryman, M., Andrady, A., Narayan, R., and Law, K.L. (2015).
Plastic waste inputs from land into the ocean.
Science 347, 768–771.
7 Napper, I.E., and Thompson, R.C. (2016).
Release of synthetic microplastic plastic fibres from domestic washing machines: Effects of fabric type and washing conditions.
Mar. Pollut. Bull. 112, 39–45.
8 Islam, S., Apitius, L., Jakob, F., and Schwaneberg, U. (2019).
Targeting microplastic particles in the void of diluted suspensions.
Environ. Int. 123, 428–435.
9 Rübsam, K., Davari, M.D., Jakob, F., and Schwaneberg, U. (2018).
KnowVolution of the polymer-binding peptide LCI for improved polypropylene binding.
Polymers (Basel). 10, 1–12.
10 Zhang, Y., Wang, L., Chen, J., and Wu, J. (2013).
Enhanced activity toward PET by site-directed mutagenesis of Thermobifida fusca cutinase-CBM fusion protein.
Carbohydr. Polym. 97, 124–129.
Our Goal
We aimed to develop a protein-made “molecular glue”, which promotes microfibers that are released from washing machines to aggregate and precipitate more easily.
Microfiber Aggregation System "SONOBE"
In order to achieve our goal, we built a system which we named "SONOBE", mainly targeting microfibers made out of PET (polyethylene terephthalate). "SONOBE" is a Japanese origami structure, whose polyhedral shape is associated with that of Encapsulins (see Gallery),so we thought it was the best for our protein device name. In this system, plastic-binding proteins are displayed on the surface of Encapsulin nanocompartments - organelle-like structures found in bacteria - which are composed of 60 Encapsulin protomers. Binding is mediated using the SpyTag-SpyCatcher system, which spontaneously forms strong covalent bonds. We engineered Encapsulin protomers to display the SpyTag protein, and each plastic-binding protein was fused with SpyCatcher. Using this approach, our engineered Encapsulins can display up to 60 SpyCatcher-fused plastic-binding proteins (Fig.1). We made use of seven plastic-binding proteins, each of which binds to different forms of plastic, such as microfibers, plastic film, or synthetic cloths, enabling the flexible use of our system. In theory, any protein which is linked with SpyCatcher can be displayed on Encapsulins, alone or in combination (Fig.2). This flexibility means that it may be applied to not only in our current project, but also other purposes as well (see Future Application).
Fig.1 The way microfibers are aggregated by protein complexes
After our proteins are introduced into laundry wastewater, they combine with microfibers through numbers of SpyCatchers and SpyTags.
Fig.2 The flexibility of the Spy-mediated bond
The advantage of Spy-system is that any SpyCatcher-fused protein can be spontaneously displayed on Encapsulins.
Our "SONOBE" protein complex can bind to microfibers through plastic-binding proteins and make them aggregate by bridging between each other, increasing their density and making it easier to precipitate. In order to introduce these proteins into wastewater, we 3D-printed a device that can be equipped with washing machines. This device is designed to provide our protein solution into laundry wastewater only when washing machines release wastewater. We envision that after these proteins are introduced into wastewater from washing machines, microfibers start to aggregate on their way to wastewater treatment plants and ultimately settle down.
Fig.3 The overview of how microfibers would be processed in our strategy
In our strategy, after proteins are introduced into laundry wastewater, microfibers would aggregate together to settle down in wastewater treatment plants, where they are removed and incinerated.