Project Overview

Why did we choose Microplastic Pollution as our target?

Having been raised by the ocean makes you realize the importance and the beauty of nature. With the worldwide industry growth, more and more plastic is produced and, one way or another, it ends up in the ocean.

Portugal's Exclusive Economic Zone (EEZ) is the 3^rd largest in the European Union, such that 11 % of the European Union's EEZ belongs to Portugal. With an area of 1,727,408 km², the Portuguese EEZ is the 5^th largest in Europe and the 20^th largest in the world [1-2]. Since Portugal really benefits from the ocean resources, it only seemed reasonable to come up with an idea to preserve this major source of income and beauty.

Concerns about the environment worldwide have been raised about the apparition of tiny plastic granules known as microplastics. The discovery of small plastic fragments in the ocean during the 1970s prompted an investigation into the potentially harmful effects of these objects in the environment, since the ingestion of microplastics could release toxins. The microplastic bioavailability benefits from its size, making them more susceptible for marine organisms’ intake [3].

Environmental Impact

Plastic pollution is already a problematic issue. Not only does it diminish and harm wildlife, but also has a negative impact on human health. More and more plastics end up in the ocean and every year 8.8 million tons of plastic enter the ocean and this number will not stop increasing. Even though there are more voluntary activities and more youngsters are aware of the plastic issue, these numbers are not decreasing. Another alarming issue caused by plastic is the accumulation of incorrectly disposed plastic in regions, causing the so-called plastic islands as the result of the ocean currents and winds [4]. After the establishment of the island, some new ecosystems are created, so when removing the island, we are destroying the newly formed life cycle. With that being said, the price of destroying that new established ecosystem is not worth the price of removing it to create a new one.

Another issue is the apparition of microplastic higher in the food chains. Meaning that microplastics that accumulate throughout the food chain, start at lower and fundamental levels such as the zooplankton. As time passes by, the microplastics accumulate in higher food chain levels and appearance in the human system is not as impossible as it would sound a few years earlier.

Consequences on the Human Health

Humanity lives in a bubble where all the problems related to pollution only lead to global warming and wildlife endangerment. However, ignorance is humanity’s worst enemy. This problem grew so big that microplastics have already entered the human organism. Scientists have studied the impact and the presence of plastic in the human organism as well as in the air we breathe.

Recent studies show that human fetuses already have traces of microplastic particles in their bodies [5], which could lead to developmental issues. These particles have also been found in lungs [6], suggesting their existence in the air; this air contamination may cause unknown harmful effects. These are only a few examples, but maybe there are even more that we are still not aware of. It’s a fruit, a vegetable, sugar or even bottled water, all the basic ingredients are already polluted and contain these tiny particles. Once they enter the human system, microplastics release toxins and other dangerous chemical substances that are foreign to our body and sometimes cannot even be metabolized. The exact effects of these materials on our health are not exactly certain, but this is not a comforting aspect since the effect may take time to be noticeable and the symptoms, as well as the consequences, may appear only many years later [7].

Economical Analysis

Microplastics are often described as a threat to our marine ecosystems and to our health. It is a known fact that marine pollution is already impacting our environmental awareness and our consumption, but its economic impacts are usually forgotten. It is estimated by the UN that the costs of plastic pollution in the oceans are more than 8 billion dollars in damages to ecosystems. Specific data on microplastics is still up for debate and as it has longer term effects it is hard to measure and to project, making the analysis rather difficult.

A quick brainstorm on how plastic pollution may have economic impacts leads to several quick conclusions. The clearest impacts are on the fishing industry and on tourism. What happens in the fishing industry is a problem that feeds upon itself. One of the major plastic polluters are the fishing boats through the waste produced by their nets and equipment. And then they suffer the consequences, they lose revenue due to the costs of repairing and replacing nets that get destroyed by plastics, due to engine and mechanical problems resulting from the entrance of garbage in their systems. A 2010 study on the Scottish fishing industry pointed out an up to 5% loss of revenues due to plastic pollution and extrapolating to the EU that might reflect on a loss of 82 million dollars per year [8]. Tourism is deeply affected as areas with higher visible or even non-visible pollution tend to be less visited and for instance a study indicated that marine litter could reduce tourism in Brazil by up to 39% and this will have economic repercussions [9].

In addition to this, one can think about the costs of removing beach litter that vary across the globe. For instance, the Netherlands and Belgium spend around 14 billion dollars per year in beach cleaning. These costs are very significant. Furthermore, an estimation by the US National Oceanic and Atmospheric Administration (NOAA) shows that only in boat time and not including labour nor equipment costs, it would cost between 122 and 489 million dollars per year to clean less than 1% of the North Pacific Ocean and bearing in mind that not all plastics would be cleaned by the equipment available, specially microplastics [10].

Once again, this reflects a dark situation with deep economic impacts that must be reversed.

On a positive note, countries and organizations are starting to raise awareness on the depth of the impacts of plastics. The European Commission, for example, has set forward the goal to address the increasing number of microplastics and this is playing a role on the European Green Deal, an initiative to tackle environmental problems. However, an EU law specifically addressing microplastics in a comprehensive way is still lacking and is currently under discussion [11].

Our Solution (Biological Design)

Our project intends to significantly reduce ocean pollution by creating a genetically modified organism (GMO) capable of degrading the major polymers found in microplastics, PE and PET respectively. To do so, our biological design relies on the innate polyethylene (PE) degradation ability of Bacillus subtilis, as our chosen chassis, and that we also aim to improve (check out how in our Project Modelling page), and the additional incorporation (by means of transformation) of the genes that code for PET degrading enzymes, PETase and MHETase, found in Ideonella sakaiensis into the B. subtilis genome as secretion enzymes. In theory, this organism would be able to incorporate PET and PE in the form of simpler metabolites and use them as a source of carbon and energy. Go to our Wet Lab page to see what we have achieved.

First, we have selected B. subtilis as our chassis strain mainly due to its capacity to degrade PE, the well-known and efficient secretion system and the availability of efficient genetic tools. To express the heterologous PET degradation pathway, the pBS1C and pBS2E plasmid backbones were used to clone our target proteins [13]. To ensure the expression of the target enzymes, the following molecular biology design was inserted into the plasmids. First, the gene expression was controlled by the promoter P43, a constitutive and strong promoter already validated for Bacillus systems, followed by a ribosome binding site (RBS). Then, since both PETase and MHETase are extracellular proteins, the immediate sequences after the RBS correspond to a signal peptide (SP) to ensure the proper secretion to the extracellular matrix. The native signal peptides of both enzymes were tested, regarding PETase an extra SP (Amy) was also implemented. Lastly, the T7 terminator sequence is downstream to the protein-coding genes completing the insert sequences.

PETase is a hydrolase, EC 3.1.1.101, with a polyethylene terephthalate degradation capability. This enzyme was first discovered in the plastic degrading bacterium I. sakaiensis as a part of a two-enzyme system, responsible for the hydrolysis of the polymer into a heterogeneous mixture of the respective constituents [14-15]. In this plasmid, it was inserted a PETase codon-optimized sequence for B. subtilis , the W159H/F229Y mutant. This mutant displays an optimal compromise between the catalysis (K_cat) and the Michaelis-Menten (K_M) constant, being 9.64 s^-1 and 0.08 mmol/L, respectively. The development of the optimized enzyme was made possible by the findings of Xiangxi’s Group [16]. This mutant together with the signal peptide, allows for the excretion of PETase by B. subtilis and a fast and efficient PET degradation inside the bioreactor.

MHETase is a hydrolase, EC 3.1.1.102, with a mono-(2-hydroxyethyl) terephthalic acid degradation capability. It is the second constituent of the two-enzyme system used by the plastic degrading bacterium I. sakaiensis to degrade PET into its constituents terephthalic acid (TPA) and ethylene glycol (EG). The MHEtase sequence inserted in this plasmid is codon-optimized for a B. subtilis chassis with a W397A mutation which displays an optimal compromise between the catalysis (K_cat) and the Michaelis-Menten (KM) constant, being 15.35 s^-1 and 1.0 μmol/L, respectively. This mutant was developed by Gottfried’s Group [17].

Lastly, we intend to incorporate PET-degrading pathway into B. subtilis, developing a commercially viable genetically engineered machine that can be deployed in a wide range of ways that we will further explore in our Proposed Implementation.

Explore P(L)AST in more Detail!

Now that you have seen the theoretical background behind our work, how about exploring P(L)AST in more detail? Click on the boxes bellow to find out more about the different sections of our work.

Lab Work

Although P(L)AST is a multi-faceted project, the biological component is its core. Above, you have read the theory behind how we designed our circuits; now, it is time to head to our WetLab page and check out our attempts at trying to make this project become a reality.

Modelling

In an effort to make our biological design even more efficient, we have spent a considerable amount of time optimizing the metabolic parameters of our chassis, trying to not only create more efficient degradation pathways but also develop a self-sustainable system.

Education & Communication

We have always kept in mind that a scientific project cannot thrive without the community that surrounds it. Spreading awareness of pressing issues, bringing the community together for a common cause or even seeking the cooperation of various organizations, you name it!

Integrated Human Practices

Being part of the scientific world comes with a good dose of humbleness. It is impossible for someone to know everything about every field. While developing our project, we embraced our relative inexperience with pride, and sought out the knowledge and expertise from people in various fields.

Proposed Implementation

P(L)AST is not meant to be just a theory project - we wanted to go further and create something practical. As such, we have focused on developing an implementation plan that is simultaneously realistic, economically efficient and easy to implement.

Multimedia

For gold criteria number 7 - excellence in another area - we decided to come up with a creative way to share our message with the masses: a photography contest, something uncommon in iGEM projects. To solidify our entry, we also developed a video series with simple and useful waste reduction practices.

References

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Portuguese Government. (2018). Zonas Marítimas sob Soberania e ou Jurisdição Portuguesa. Retrieved on 12-10-2021 from https://www.dgrm.mm.gov.pt/am-ec-zonas-maritimas-sob-jurisdicao-ou-soberania-nacional
Cole, M., Lindeque, P., Halsband, C., & Galloway, T. S. (2011). Microplastics as contaminants in the marine environment: A review. Marine Pollution Bulletin, 62(12), 2588–2597. https://doi.org/10.1016/j.marpolbul.2011.09.025
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J. Mouat, R.L. Lozano, H. Bateson, Economic impacts of marine litter, Kommunenes Internasjonale Miljøorganisasjon, Lerwick, 2010.
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Maity, W., Maity, S., Bera, S., & Roy, A. (2021). Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes. Applied Biochemistry and Biotechnology, 1-18.
Meng, X., Yang, L., Liu, H., Li, Q., Xu, G., Zhang, Y., ... & Tian, J. (2021). Protein engineering of stable IsPETase for PET plastic degradation by Premuse. International Journal of Biological Macromolecules, 180, 667-676.
Palm, G. J., Reisky, L., Böttcher, D., Müller, H., Michels, E. A., Walczak, M. C., ... & Weber, G. (2019). Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate. Nature communications, 10(1), 1-10.