Difference between revisions of "Team:HK GTC/Description"

 
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        <h1>Project Description</h1>
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      <h1>Project Description</h1>
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        <h1>Project Description</h1>
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        <li><a href="#1">Why do we choose PET plastic?</a></li>
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        <ul>
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        <li><a href="#2">What is the dual-enzyme system for PET depolymerization?</a></li>
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            <li><a href="#current">Current situation of plastic pollution</a></li>
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        <li><a href="#3">How can the dual enzyme system synergize PET depolymerization process?</a></li>
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            <li><a href="#inst">Inspiration</a></li>
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        <li><a href="#4">How do we develop the dual enzyme system of PET depolymerization?</a></li>
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            <li><a href="#sol">Solution</a></li>
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        <li><a href="#5">Our goal</a></li>
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            <li><a href="#nase">PETase and its mutant, S245I</a></li>
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        <li><a href="#6">References </a></li>
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            <li><a href="#chrim">Chimeric proteins and enzyme cocktails</a></li>
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      </ul>
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            <li><a href="#result">Results</a></li>
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        <p class="intro">The global plastic problem has been a widely discussed issue. After the invention of
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            <h2 id="current">Current situation of plastic pollution</h2>
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            polyethylene
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            <p>Global plastic problem has been a widely discussed issue. After the invention of polyethylene terephthalate
+
            terephthalate (PET) plastic bottles in 1973, the global PET plastic production has risen dramatically. PET
−
              (PET) plastic bottles in 1973, the global PET plastic production has risen dramatically. PET bottles are
+
            bottles are known for their strength and for their durability. Unfortunately, the convenience of using PET
−
              known for their strength and for their durability. Unfortunately, the convenience of using PET bottles comes
+
            bottles comes at a cost. More than 360 million tonnes of plastic waste is produced annually worldwide [1],
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              at a cost. More than 360 million tonnes of plastic waste is produced annually worldwide [1], and an
+
            and an estimated amount of 5.25 trillion pieces of plastic and microplastic are currently floating around
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              estimated amount of 5.25 trillion pieces of plastic and microplastic are currently floating around the ocean
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            the ocean [2]. By 2050, it is estimated that more plastic than fish will be filling up our oceans [3]. Our
−
              [2]. By 2050, it is estimated that more plastic than fish will be filling up our oceans [3]. Our team,
+
            team, HK_GTC, notices the severity of plastic pollution, and is dedicated to developing solutions to solve
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              HK_GTC, notices the severity of plastic pollution, and is dedicated to solving the global problem of plastic
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            the global problem of plastic pollution and arousing public awareness of this issue.
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              pollution and arousing public awareness to this issue.</p>
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        </p>
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            <h2 id="ins">Inspiration</h2>
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        <h2 id="1">Why do we choose PET plastic?</h2>
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            <p>We believe that PET plastic is the main contributing factor of global plastic pollution. PET contributes 94%
+
        <p>We believe that PET plastic is the main contributing factor of global plastic pollution. PET contributes 93%
−
              by weight in a plastic bottle [4], and it contributed to 20% of global plastic production in 2020 [5]. To
+
            by weight in a plastic bottle [4]. It also contributes to 20% of global plastic production in 2020 [5][6].
−
              ease the plastic pollution problem, our project focuses on completely depolymerizing PET plastics into its
+
        </p>
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              constituting monomers. In 2016, a species of PET-digesting bacteria, Ideonella sakaiensis, was discovered in
+
 
−
              a Japanese recycling plant located in Sakai. This bacteria was found to secrete PETase and MHETase. We see
+
        <h2 id="2">What is the dual-enzyme system for PET depolymerization?</h2>
−
              that a dual-enzyme system consisting of PETase and MHETase can digest PET in nature. Therefore we
+
        <p>In 2016, a species of PET-digesting bacterium, <i>Ideonella sakaiensis</i>, was discovered in a Japanese recycling
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              hypothesize that this dual-enzyme system will also be capable of digesting PET plastics in our project.</p>
+
            plant located in Sakai. This bacterium was found to secrete PETase and MHETase, in which PETase is capable
−
 
+
            of hydrolysing PET into reaction intermediates, and MHETase capable of digesting the reaction intermediates
−
            <h2 id="sol">Solution</h2>
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            into the constituting monomers of PET [7]. We see that nature is evolving to depolymerize PET plastics with
−
            <p>
+
            a dual-enzyme system. Therefore we hypothesized that this dual-enzyme system is also capable of degrading
−
              The ultimate goal of this project is to use a protein engineering approach to develop a dual-enzyme system
+
            PET plastics in our project.</p>
−
              in which two enzymes act synergistically to completely degrade PET into its constituting monomers. These
+
 
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              monomers can be further synthesized back into PET and other useful products, allowing the plastic industry
+
        <h2 id="3">How can the dual enzyme system synergize PET depolymerization process?</h2>
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              to develop more sustainably.
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        <p>Two enzymes, PETase and MHETase, are used to depolymerize PET. Using PETase, PET is first broken down into
−
 
+
            three monomers: bis(2-hydroxyethyl) terephthalic acid (BHET), mono(2-hydroxyethyl) terephthalic acid (MHET),
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              We propose the usage of two enzymes, PETase and MHETase, to depolymerize PET. Using PETase, PET is first
+
            and terephthalic acid (TPA). MHETase further catalyzes the breakdown of MHET into TPA and ethylene glycol
−
              broken down into three monomers: bis(2-hydroxyethyl) terephthalic acid (BHET), mono(2-hydroxyethyl)
+
            (EG) (Figure 1).</p>
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              terephthalic acid (MHET), and terephthalic acid (TPA). MHETase further catalyzes the breakdown of MHET into
+
 
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              TPA and ethylene glycol (EG) (Figure 1).
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        <div class="single-image-with-desc">
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            <center><img src="https://static.igem.org/mediawiki/2021/2/20/T--HK_GTC--pd1.png" alt=""></center>
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            <p>Figure 1. The breakdown process of PET using PETase and MHETase.
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            <div class="single-image-with-desc">
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              Left: PETase depolymerizes PET into BHET, MHET and TPA.
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              <center><img src="../img/1.png" alt=""></center>
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              Right: MHETase depolymerizes MHET into TPA and EG.</p>
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              <p>Figure 1. The breakdown process of PETing usPETase and MHETase. Left: PETase depolymerizes PET into BHET,
+
        </div>
−
                  MHET and TPA. Right: MHETase depolymerizes MHET into TPA and EG.</p>
+
 
 +
        <p>
 +
            In 2019, our team created two successful PETase mutants that can increase the enzymatic activity of WT
 +
            PETase. This year, we perform PET film digestion and High Performance Liquid Chromatography (HPLC) of PET
 +
            digestion eluent to study the effect of PETase and S245I on PET degradation. This is to confirm the
 +
            performance of our engineered mutant. Both the Scanning Electron Microscope (SEM) images and quantification
 +
            of reaction products by HPLC indicate that our engineered mutant, S245I outperforms the wild type PETase, as
 +
            seen by the increased surface erosion of PET film and the increased release of monomeric products. HPLC
 +
            profiles of products released from PET film digestions reveal considerable amounts of intermediate product,
 +
            MHET, suggesting that PET cannot be completely depolymerized by PETase alone (Figure 2).
 +
            <br><br>
 +
            Thus, we hypothesized that the presence of a second enzyme, MHETase, in the PET depolymerization system
 +
            synergizes the degradation rate of PET into its constituting monomers.
 +
        </p>
 +
 
 +
        <div class="two-image-with-desc">
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            <div class="im-group">
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              <center><img src="https://static.igem.org/mediawiki/2021/5/52/T--HK_GTC--pd3.png" alt=""></center>
 +
              <center><img src="https://static.igem.org/mediawiki/2021/c/cf/T--HK_GTC--pd2.png" alt=""></center>
 
             </div>
 
             </div>
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             <p>Figure 2. HPLC data of the products obtained after 96 hours of PET digestion at 30°C.
−
            <h2 id="nase">PETase and its mutant, S245I</h2>
+
               The retention time of TPA and MHET HPLC standards were at 4.64 minutes and 5.17 minutes respectively.
−
             <p>In 2019, our team created two successful PETase mutants that can increase the enzymatic activity of PETase.
+
               Left: PET film digestion using wild type PETase as the only enzyme.
−
               The single-mutant S245I, and the double-mutant W159H/S245I proved to have a higher depolymerization activity
+
               Right: PET film digestion using S245I PETase as the only enzyme.
−
              as compared with the wild type. This year, we did some follow-up experiments to confirm if our PETase mutant
+
−
              S245I can successfully digest PET. We used a Scanning Electron Microscope (SEM) to observe the pitting of
+
−
               the digested PET film surface. The HPLC result shows the levels of the intermediate product of digestion,
+
−
              MHET, and the monomer, TPA.
+
−
 
+
−
              We showed that when only wild type PETase and the S245I PETase is present in the digestion process, MHET was
+
−
               detected, which suggests that PET is not completely depolymerized by PETase (Table 1). Therefore, we
+
−
              hypothesize the presence of MHETase in our enzyme system can increase the degradation rate of PET into its
+
−
              constituting monomers.
+
 
             </p>
 
             </p>
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            <div class="two-image-with-desc">
 
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              <div class="im-group">
 
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                  <center><img src="../img/2.png" alt=""></center>
 
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                  <center><img src="../img/3.png" alt=""></center>
 
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              </div>
 
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              <p>Figure 2. HPLC data of the products obtained after 96 hours of PET digestion at 30°C.
 
−
                  The retention time of TPA and MHET HPLC standards were at 4.64 minutes and 5.17 minutes respectively.
 
−
                  Left: PETase digestion using wild type PETase as the only enzyme.
 
−
                  Right: PETase digestion using S245I PETase as the only enzyme.
 
−
              </p>
 
−
            </div>
 
−
 
 
−
            <h2 id="chrim">Chimeric proteins and enzyme cocktails</h2>
 
−
            <p>We hypothesize that adding MHETase with PETase will synergize the PET depolymerization process. We propose
 
−
              to develop a dual-enzyme of PETase and MHETase system in forms of chimeric proteins and enzyme cocktails.
 
−
              For the chimeric proteins of PETase (including PETase mutants) and MHETase, we link the C-terminus of PETase
 
−
              to the N-terminus of MHETase using a 12 amino acid serine-glycine linker. We expect the efficiency of the
 
−
              degradation of PET into its final monomers, TPA and EG, will be increased. For the protein cocktails of
 
−
              PETase (including PETase mutants) and MHETase, we mix PETase with MHETase in a single reaction. We would
 
−
              also like to compare the depolymerization activities of the chimeric protein and the protein cocktail.</p>
 
−
 
 
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            <h2 id="result">Results</h2>
 
−
            <p>In the start of our project, we designed our new constructs, cut using the restriction enzyme and did PCR
 
−
              screening. Following that, we did protein induction to express the protein, and protein extraction and
 
−
              purification. We did Bradford protein assay to test the concentration of protein, and SDS-PAGE to confirm if
 
−
              our protein is expressed. Finally, we did PET film digestion and used HPLC and SEM equipment borrowed from
 
−
              HKU to analyse the results of the experiment (See Figure 3).
 
−
            </p>
 
−
 
 
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            <h2 id="result">Detection of Plastic Bottles</h2>
 
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            <p>
 
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            </p>
 
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            <p>
 
−
              References <br>
 
−
              [1]: “EU plastics production and demand first estimates for 2020” <br>
 
−
              [2]: ?<br>
 
−
              [3]: The New Plastics Economy, Rethinking The Future of Plastics (Rep.). (2016). Geneva, Switzerland: The
 
−
              World Economic Forum. <br>
 
−
              [4]: <br>
 
−
              [5]: <br>
 
−
              [6]: Austin, H. P., Allen, M. D. et al. (2018). Characterization and engineering of a plastic-degrading
 
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              aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19).
 
−
              doi:10.1073/pnas.1718804115<br>
 
−
              [7]: Yoshida, S., Hiraga, K. et al. (2016). A bacterium that degrades and assimilates poly(ethylene
 
−
              terephthalate). Science,351(6278), 1196-1199. doi:10.1126/science.aad6359 <br>
 
−
              [8]: Knott, B. C., Erickson, E. et al. (2020). Characterization and engineering of a two-enzyme system for
 
−
              plastics depolymerization, pnas.org. doi:10.1073/pnas.2006753117<br>
 
−
              [9]: Han, X., Liu, W. et al. (2017). Structural insight into catalytic mechanism of PET hydrolase, nature
 
−
              communications. DOI:10.1038/s41467-017-02255-z<br>
 
−
              [10]: Joo, S., Cho, I. J. et al. (2018). Structural insight into molecular mechanism of poly(ethylene
 
−
              terephthalate) degradation. Nature Communications, 9(1). doi:10.1038/s41467-018-02881-<br>
 
−
            </p>
 
−
 
 
 
         </div>
 
         </div>
 +
 +
 +
 +
        <h2 id="4">How do we develop the dual enzyme system of PET depolymerization?</h2>
 +
        <p>In our project, we synergize the PET depolymerization process by adding MHETase with PETase. A dual-enzyme
 +
            system of PETase and MHETase is developed in the form of either as chimeras or enzyme cocktails. For the
 +
            chimeric proteins of PETase (including PETase mutant, S245I) and MHETase, we link the C-terminus of PETase
 +
            to the N-terminus of MHETase using a 12 amino acid serine-glycine linker. For the enzyme cocktails of PETase
 +
            (including a PETase mutant, S245I) and MHETase, PETase is mixed with MHETase in a single reaction. We
 +
            compare the depolymerization activities of the chimeric protein and the enzyme cocktail.
 +
        </p>
 +
 +
        <h2 id="5">Our goal</h2>
 +
        <p>The ultimate goal of our project is to use a protein engineering approach to develop a dual-enzyme system,
 +
            in which two enzymes act synergistically to completely degrade PET into its constituting monomers. These
 +
            monomers can be further synthesized back into PET, reducing the reliance on fossil fuels and allowing the
 +
            plastic industry to develop more sustainably. We also hope that by combining PETase with a second enzyme,
 +
            MHETase, the PET degradation process will be fast enough to handle tonnes of PET plastics wastes drowning
 +
            the planet.</p>
 +
 +
        <video width="640" height="480" controls>
 +
            <source src="https://static.igem.org/mediawiki/2021/2/2d/T--HK_GTC--promo_vid.mp4" type="video/mp4">
 +
        </video>
 +
 +
        <h2 id="6">References</h2>
 +
        <p>
 +
            [1]: “EU plastics production and demand first estimates for 2020”,
 +
            https://www.plasticseurope.org/en/newsroom/news/eu-plastics-production-and-demand-first-estimates-2020
 +
            <br>
 +
            [2]: National Geographic Society. (2019, February 22). Ocean Trash: 5.25 Trillion Pieces and Counting, but
 +
            Big Questions Remain.
 +
            https://www.nationalgeographic.org/article/ocean-trash-525-trillion-pieces-and-counting-big-questions-remain/
 +
            <br>
 +
            [3]: The New Plastics Economy, Rethinking The Future of Plastics (Rep.). (2016). Geneva, Switzerland: The
 +
            World Economic Forum.
 +
            <br>
 +
            [4]: PET & HDPE. (2020, November 23). New Life Plastics. https://www.nlplastics.com.hk/pet-hdpe/
 +
            <br>
 +
            [5]: Statista. (2021, September 10). Global plastic production 1950–2020.
 +
            https://www.statista.com/statistics/282732/global-production-of-plastics-since-1950/
 +
            <br>
 +
            [6]: Statista. (2021a, January 27). Global polyethylene terephthalate production 2014–2020.
 +
            https://www.statista.com/statistics/650191/global-polyethylene-terephthalate-production-outlook/
 +
            <br>
 +
            [7]: Yoshida, S., Hiraga, K. et al. (2016). A bacterium that degrades and assimilates poly(ethylene
 +
            terephthalate). Science,351(6278), 1196-1199. doi:10.1126/science.aad6359
 +
 +
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Latest revision as of 15:04, 19 October 2021

HK_GTC 2021 Homepage

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Project Description

Project Description

The global plastic problem has been a widely discussed issue. After the invention of polyethylene terephthalate (PET) plastic bottles in 1973, the global PET plastic production has risen dramatically. PET bottles are known for their strength and for their durability. Unfortunately, the convenience of using PET bottles comes at a cost. More than 360 million tonnes of plastic waste is produced annually worldwide [1], and an estimated amount of 5.25 trillion pieces of plastic and microplastic are currently floating around the ocean [2]. By 2050, it is estimated that more plastic than fish will be filling up our oceans [3]. Our team, HK_GTC, notices the severity of plastic pollution, and is dedicated to developing solutions to solve the global problem of plastic pollution and arousing public awareness of this issue.

Why do we choose PET plastic?

We believe that PET plastic is the main contributing factor of global plastic pollution. PET contributes 93% by weight in a plastic bottle [4]. It also contributes to 20% of global plastic production in 2020 [5][6].

What is the dual-enzyme system for PET depolymerization?

In 2016, a species of PET-digesting bacterium, Ideonella sakaiensis, was discovered in a Japanese recycling plant located in Sakai. This bacterium was found to secrete PETase and MHETase, in which PETase is capable of hydrolysing PET into reaction intermediates, and MHETase capable of digesting the reaction intermediates into the constituting monomers of PET [7]. We see that nature is evolving to depolymerize PET plastics with a dual-enzyme system. Therefore we hypothesized that this dual-enzyme system is also capable of degrading PET plastics in our project.

How can the dual enzyme system synergize PET depolymerization process?

Two enzymes, PETase and MHETase, are used to depolymerize PET. Using PETase, PET is first broken down into three monomers: bis(2-hydroxyethyl) terephthalic acid (BHET), mono(2-hydroxyethyl) terephthalic acid (MHET), and terephthalic acid (TPA). MHETase further catalyzes the breakdown of MHET into TPA and ethylene glycol (EG) (Figure 1).

Figure 1. The breakdown process of PET using PETase and MHETase. Left: PETase depolymerizes PET into BHET, MHET and TPA. Right: MHETase depolymerizes MHET into TPA and EG.

In 2019, our team created two successful PETase mutants that can increase the enzymatic activity of WT PETase. This year, we perform PET film digestion and High Performance Liquid Chromatography (HPLC) of PET digestion eluent to study the effect of PETase and S245I on PET degradation. This is to confirm the performance of our engineered mutant. Both the Scanning Electron Microscope (SEM) images and quantification of reaction products by HPLC indicate that our engineered mutant, S245I outperforms the wild type PETase, as seen by the increased surface erosion of PET film and the increased release of monomeric products. HPLC profiles of products released from PET film digestions reveal considerable amounts of intermediate product, MHET, suggesting that PET cannot be completely depolymerized by PETase alone (Figure 2).

Thus, we hypothesized that the presence of a second enzyme, MHETase, in the PET depolymerization system synergizes the degradation rate of PET into its constituting monomers.

Figure 2. HPLC data of the products obtained after 96 hours of PET digestion at 30°C. The retention time of TPA and MHET HPLC standards were at 4.64 minutes and 5.17 minutes respectively. Left: PET film digestion using wild type PETase as the only enzyme. Right: PET film digestion using S245I PETase as the only enzyme.

How do we develop the dual enzyme system of PET depolymerization?

In our project, we synergize the PET depolymerization process by adding MHETase with PETase. A dual-enzyme system of PETase and MHETase is developed in the form of either as chimeras or enzyme cocktails. For the chimeric proteins of PETase (including PETase mutant, S245I) and MHETase, we link the C-terminus of PETase to the N-terminus of MHETase using a 12 amino acid serine-glycine linker. For the enzyme cocktails of PETase (including a PETase mutant, S245I) and MHETase, PETase is mixed with MHETase in a single reaction. We compare the depolymerization activities of the chimeric protein and the enzyme cocktail.

Our goal

The ultimate goal of our project is to use a protein engineering approach to develop a dual-enzyme system, in which two enzymes act synergistically to completely degrade PET into its constituting monomers. These monomers can be further synthesized back into PET, reducing the reliance on fossil fuels and allowing the plastic industry to develop more sustainably. We also hope that by combining PETase with a second enzyme, MHETase, the PET degradation process will be fast enough to handle tonnes of PET plastics wastes drowning the planet.

References

[1]: “EU plastics production and demand first estimates for 2020”, https://www.plasticseurope.org/en/newsroom/news/eu-plastics-production-and-demand-first-estimates-2020
[2]: National Geographic Society. (2019, February 22). Ocean Trash: 5.25 Trillion Pieces and Counting, but Big Questions Remain. https://www.nationalgeographic.org/article/ocean-trash-525-trillion-pieces-and-counting-big-questions-remain/
[3]: The New Plastics Economy, Rethinking The Future of Plastics (Rep.). (2016). Geneva, Switzerland: The World Economic Forum.
[4]: PET & HDPE. (2020, November 23). New Life Plastics. https://www.nlplastics.com.hk/pet-hdpe/
[5]: Statista. (2021, September 10). Global plastic production 1950–2020. https://www.statista.com/statistics/282732/global-production-of-plastics-since-1950/
[6]: Statista. (2021a, January 27). Global polyethylene terephthalate production 2014–2020. https://www.statista.com/statistics/650191/global-polyethylene-terephthalate-production-outlook/
[7]: Yoshida, S., Hiraga, K. et al. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). Science,351(6278), 1196-1199. doi:10.1126/science.aad6359

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