Difference between revisions of "Team:RHIT/Design"

 
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<div class = "column full_size">
 
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<a href = "https://2018.igem.org/Team:RHIT/Description">
 
<a href = "https://2018.igem.org/Team:RHIT/Description">
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   <img id="dsc" src = "https://static.igem.org/mediawiki/2018/2/24/T--RHIT--DescriptionSubMenuButton.png" style="width:150px"> </a>
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   <img id="dsc" src="https://static.igem.org/mediawiki/2018/d/da/T--RHIT--petrihover.jpg" style="width:140px">
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      <h6 style="font-size:100%" id="dsct"> Description </h6> </a>
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    <img id="des" src = "https://static.igem.org/mediawiki/2018/4/47/T--RHIT--petri.jpg" style="width:140px">
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      <h6 style="font-size:100%" id="dest"> Design </h6>
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<a href="https://2018.igem.org/Team:RHIT/Experiments">
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      <h6 style="font-size:100%" id="expt"> Experiments </h6></a>
  
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<a href="https://2018.igem.org/Team:RHIT/Experiments">
 
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<a href="https://2018.igem.org/Team:RHIT/Notebook">  
 
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      <h6 style="font-size:100%" id="notet"> Notebook </h6></a>
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<a href="https://2018.igem.org/Team:RHIT/Results">  
 
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      <h6 style="font-size:100%" id="rest"> Results </h6></a>
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<a href="https://2018.igem.org/Team:RHIT/Achievements">  
 
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        <h6 style="font-size:100%" id="acht"> Achievements </h6></a>
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<a href="https://2018.igem.org/Team:RHIT/InterLab">  
 
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<a href="https://2018.igem.org/Team:RHIT/Attributions">  
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      <h6 style="font-size:100%" id="inlt"> InterLab </h6> </a>
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<a href="https://2018.igem.org/Team:RHIT/Attributions">
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  <img id="attr" src="https://static.igem.org/mediawiki/2018/d/da/T--RHIT--petrihover.jpg" style="width:140px">  
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<h6 style="font-size:100%" id="attrt"> Attributions </h6> </a>
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<div class="column full_size">
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<h1>Design</h1>
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<h5> Our Design </h5>
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<p>
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<p>For our project, we have designed a plasmid that secretes MHETase and the double mutant PETase to increase the rate at which PET is degraded compared to the previous PETase sequence. We inserted the plasmid into an <em>E. coli </em>MG1655 strain. Because of the toxicity of ethylene glycol, a second plasmid was designed to allow the bacteria to break down the ethylene glycol and utilize its products as a carbon source. These enzymes include glycolaldehyde reductase, glycolaldehyde dehydrogenase, glycolate oxidase, and malate synthase. This series of enzymes will turn the ethylene glycol, released from the breakdown of PET, into malate which can be used by the cell as a carbon source via the citric acid cycle. <br><br>
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Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
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</p>
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<p>
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Our <em>E. coli</em> will degrade PET into terephthalic acid (TPA)  and ethylene glycol (EG) utilizing the PETase and MHETase enzymes, as shown in Figure 1 below. </p>
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This page is different to the "Applied Design Award" page. Please see the <a href="https://2018.igem.org/Team:RHIT/Applied_Design">Applied Design</a> page for more information on how to compete for that award.
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<center>
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</p>
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<img src = "https://static.igem.org/mediawiki/2018/9/9d/T--RHIT--design1.jpg" style="width:80%">
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</center>
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<center> Figure 1. Initial breakdown of PET chains to byproducts Ethylene Glycol and Terephthalic acid. </center>
 +
<br><br>
  
−
</div>
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<p>Terephthalic acid is recyclable when purified and is relatively nontoxic. Ethylene glycol, on the other hand, is toxic when ingested by humans, although it has been proven to be safe for <em>E. coli </em>cells.
 +
<br><br>
 +
The <em>E. coli</em> will then take the ethylene glycol and utilize it as a carbon source using glycolaldehyde reductase, glycolaldehyde dehydrogenase, glycolate oxidase, and malate synthase. This will result in intermediates of glycolaldehyde, glycolate, and glyoxylate, as well as the product of malate which will be used in the citric acid cycle, as shown in Figure 2 below. </p>
 +
<center>
 +
<img src = "https://static.igem.org/mediawiki/2018/3/30/T--RHIT--design2_.jpg">
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</center>
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<center> Figure 2. Further breakdown of Ethylene Glycol to Malate. </center>
 +
<br><br>
  
 +
<p>Two plasmids were designed to accomplish our goals. The first plasmid (Figure 3) contains PETase and MHETase, as well as the pelB secretion tag. The second plasmid (Figure 4) contains the enzyme series for the breakdown of ethylene glycol. </p>
  
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<h3>What should this page contain?</h3>
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<center>
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<ul>
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<img src = "https://static.igem.org/mediawiki/2018/e/ec/T--RHIT--plasmid1.jpg">
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<li>Explanation of the engineering principles your team used in your design</li>
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</center>
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<li>Discussion of the design iterations your team went through</li>
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<center> Figure 3. </center>
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<li>Experimental plan to test your designs</li>
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</div>
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</ul>
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<div class = "column half_size">
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<center>
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<img src = "https://static.igem.org/mediawiki/2018/c/c9/T--RHIT--plasmid2.jpg">
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</center>
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<center> Figure 4 </center>
 
</div>
 
</div>
  
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<div class="column third_size">
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<div class = "column full_size">
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<div class="highlight decoration_A_full">
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<br><br>
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<h3>Inspiration</h3>
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<p> Our team selected the W159H/S238F double mutated enzyme based on the article released in April of 2018. The article analyzed the structure of PETase to design the specific double amino acid mutation. PETase was found to have similar common features to cutinases and lipases, which are also shown to partially degrade PET. The double mutation changes the PETase structure by narrowing the binding cleft to better resemble cutinase. In Figure 5 below, which comes from the article, E shows the proposed structure of the binding site in wild-type PETase, while F shows the proposed structure of the binding site in the W159H/S238F double mutated PETase [1]. </p>
 +
 
 +
<center>
 +
<img src = "https://static.igem.org/mediawiki/2018/2/2e/T--RHIT--doublemutant.jpg" style="width:70%">
 +
</center>
 +
<center> Figure 5 Ball and Stick representation of the double mutated PETase chain from the article [1].</center>
 +
 
 +
<br><br>
 +
 
 +
<h5> References </h5>
 
<ul>
 
<ul>
−
<li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
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<li>[1] Austin, H., Allen, M., Donohoe, B., Rorrer, N., Kearns, F., Silveira, R., Pollard, B., Dominick, G., Duman, R., El Omari, K., Mykhaylyk, V., Wagner, A., Michener, W., Amore, A., Skaf, M., Crowley, M., Thorne, A., Johnson, C., Woodcock, H., McGeehan, J. and Beckham, G. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19), pp.E4350-E4357.</li>
−
<li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
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−
<li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li>
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Latest revision as of 02:47, 13 October 2018




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Description
Design
Experiments
Notebook
Results
Achievements
InterLab
Attributions
Our Design

For our project, we have designed a plasmid that secretes MHETase and the double mutant PETase to increase the rate at which PET is degraded compared to the previous PETase sequence. We inserted the plasmid into an E. coli MG1655 strain. Because of the toxicity of ethylene glycol, a second plasmid was designed to allow the bacteria to break down the ethylene glycol and utilize its products as a carbon source. These enzymes include glycolaldehyde reductase, glycolaldehyde dehydrogenase, glycolate oxidase, and malate synthase. This series of enzymes will turn the ethylene glycol, released from the breakdown of PET, into malate which can be used by the cell as a carbon source via the citric acid cycle.

Our E. coli will degrade PET into terephthalic acid (TPA) and ethylene glycol (EG) utilizing the PETase and MHETase enzymes, as shown in Figure 1 below.

Figure 1. Initial breakdown of PET chains to byproducts Ethylene Glycol and Terephthalic acid.


Terephthalic acid is recyclable when purified and is relatively nontoxic. Ethylene glycol, on the other hand, is toxic when ingested by humans, although it has been proven to be safe for E. coli cells.

The E. coli will then take the ethylene glycol and utilize it as a carbon source using glycolaldehyde reductase, glycolaldehyde dehydrogenase, glycolate oxidase, and malate synthase. This will result in intermediates of glycolaldehyde, glycolate, and glyoxylate, as well as the product of malate which will be used in the citric acid cycle, as shown in Figure 2 below.

Figure 2. Further breakdown of Ethylene Glycol to Malate.


Two plasmids were designed to accomplish our goals. The first plasmid (Figure 3) contains PETase and MHETase, as well as the pelB secretion tag. The second plasmid (Figure 4) contains the enzyme series for the breakdown of ethylene glycol.

Figure 3.
Figure 4


Our team selected the W159H/S238F double mutated enzyme based on the article released in April of 2018. The article analyzed the structure of PETase to design the specific double amino acid mutation. PETase was found to have similar common features to cutinases and lipases, which are also shown to partially degrade PET. The double mutation changes the PETase structure by narrowing the binding cleft to better resemble cutinase. In Figure 5 below, which comes from the article, E shows the proposed structure of the binding site in wild-type PETase, while F shows the proposed structure of the binding site in the W159H/S238F double mutated PETase [1].

Figure 5 Ball and Stick representation of the double mutated PETase chain from the article [1].


References
  • [1] Austin, H., Allen, M., Donohoe, B., Rorrer, N., Kearns, F., Silveira, R., Pollard, B., Dominick, G., Duman, R., El Omari, K., Mykhaylyk, V., Wagner, A., Michener, W., Amore, A., Skaf, M., Crowley, M., Thorne, A., Johnson, C., Woodcock, H., McGeehan, J. and Beckham, G. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19), pp.E4350-E4357.