Difference between revisions of "Team:UMaryland/Improve"

 
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<div class="titleText">PET NET</div>
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<div class="titleText">Improve</div>
 
<div class="subtitleText">Increasing the Efficacy of PETase</div>
 
<div class="subtitleText">Increasing the Efficacy of PETase</div>
 
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One of the factors that affects the efficiency of plastic biodegradation is the concentration of degrading enzyme near the surface of plastic. If the enzyme is allowed to diffuse in solution freely, it is less likely to interact with plastic. Degradation may accelerate if the enzyme is highly concentrated around the surface of plastic with some method. The bacterium Ideonella sakaiensis was reported to have grown in a biofilm which enveloped plastic pieces. As such, we first considered maximizing production of biofilm on plastic. While researching this possibility we found that the <a href="https://2017.igem.org/Team:ITB_Indonesia"><u>ITB Indonesia 2017</u></a> had attempted a similar project. They developed a system that facilitated biofilm production and expressed PETase simultaneously. Their results indicated that biofilm formation actually inhibited degradation, possibly due to its matrix physically blocking the transfer of enzymes to plastic.
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One of the factors that affects the efficiency of biodegradation of plastics is the concentration of the catalyzing enzyme near the surface of the plastic. If the enzyme is allowed to diffuse in solution freely it is less likely to interact with the plastic. In a situation like this degradation could be much slower than if the enzyme was highly concentrated around the surface of the plastic. The Ideonella sakaiensis was reported to have grown in a biofilm which enveloped plastic pieces. As such, we first considered maximizing production of biofilm on plastic. While researching this possibility we found that the 2017 ITB Indonesia iGEM team had attempted a similar project. They developed a system that facilitated biofilm production and expressed PETase simultaneously. Their results indicated that biofilm formation actually inhibited degradation. More plastic was degraded without biofilm than with.
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                                                      <img src="https://static.igem.org/mediawiki/2018/2/22/T--UMaryland--cellulosePETase.png" style="max-width: 80%" alt="Waluigi Time!">
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We decided to investigate other methods of localizing concentration while simultaneously allowing for the greater interaction of the substrate and enzyme than biofilm was seen to do. Imperial College of London in 2014 developed parts for a cellulose binding domain (CBD). This domain was capable of being incorporated with other subunits such as GFP. They were able to demonstrate that a GFP + CBD fusion protein was capable of binding to bacterial cellulose. Equipped with this information we decided to develop a fusion protein which incorporated PETase and CBD. Our hypothesis is that the bacterial cellulose surrounding the plastic will act as a scaffold on which the PETase can easily work in close proximity to the substrate. This should, in turn, increase the degradation rate. A model of our predictions can be found on the modeling page.
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<div class="imageBoxDescription">Figure 1 - Float right Waluigi time!</div>
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<img src="https://static.igem.org/mediawiki/2018/2/22/T--UMaryland--cellulosePETase.png" style="max-width: 100%" alt="Waluigi Time!">
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Waluigi is the ultimate example of the individual shaped by the signifier. Waluigi is a man seen only in mirror images; lost in a hall of mirrors he is a reflection of a reflection of a reflection. You start with Mario – the wholesome all Italian plumbing superman, you reflect him to create Luigi – the same thing but slightly less. You invert Mario to create Wario – Mario turned septic and libertarian – then you reflect the inversion in the reflection: you create a being who can only exist in reference to others. Waluigi is the true nowhere man, without the other characters he reflects, inverts and parodies he has no reason to exist. Waluigi’s identity only comes from what and who he isn’t – without a wider frame of reference he is nothing. He is not his own man. In a world where our identities are shaped by our warped relationships to brands and commerce we are all Waluigi.
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We therefore decided to investigate an alternative method for localizing concentration while simultaneously allowing for greater interaction of substrate with enzyme. <a href="https://2014.igem.org/Team:Imperial"><u>Imperial College of London in 2014</u></a> developed parts for a cellulose binding domain (CBD). This domain was capable of being incorporated with other subunits such as GFP. They were able to demonstrate that a GFP + CBD fusion protein was capable of binding to bacterial cellulose. Equipped with this information, we decided to develop a fusion protein which incorporated PETase and CBD. We assembled this part, and its characterization is available on the <a href="http://parts.igem.org/Part:BBa_K2825000"><u>registry</u></a>. We have tested the functionality of both its PETase component and its cellulose binding component. The part also includes an HlyA secretion sytem, but we were unable to confirm secretion.
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Our hypothesis is that we can wrap bacterial cellulose around plastic to act as a scaffold on which the PETase can easily work in close proximity to the substrate. This should, in turn, increase the degradation rate. A <a href="https://2018.igem.org/Team:UMaryland/BCmodel"><u>model of our predictions</u></a> can be found on the modeling page.
 
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<img src="https://static.igem.org/mediawiki/2018/7/77/T--UMaryland--waaaa.png" style="max-width: 100%" alt="Waluigi Time!">
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Latest revision as of 01:47, 18 October 2018

Template Title Template Title

Improve
Increasing the Efficacy of PETase
One of the factors that affects the efficiency of plastic biodegradation is the concentration of degrading enzyme near the surface of plastic. If the enzyme is allowed to diffuse in solution freely, it is less likely to interact with plastic. Degradation may accelerate if the enzyme is highly concentrated around the surface of plastic with some method. The bacterium Ideonella sakaiensis was reported to have grown in a biofilm which enveloped plastic pieces. As such, we first considered maximizing production of biofilm on plastic. While researching this possibility we found that the ITB Indonesia 2017 had attempted a similar project. They developed a system that facilitated biofilm production and expressed PETase simultaneously. Their results indicated that biofilm formation actually inhibited degradation, possibly due to its matrix physically blocking the transfer of enzymes to plastic.
Waluigi Time!
We therefore decided to investigate an alternative method for localizing concentration while simultaneously allowing for greater interaction of substrate with enzyme. Imperial College of London in 2014 developed parts for a cellulose binding domain (CBD). This domain was capable of being incorporated with other subunits such as GFP. They were able to demonstrate that a GFP + CBD fusion protein was capable of binding to bacterial cellulose. Equipped with this information, we decided to develop a fusion protein which incorporated PETase and CBD. We assembled this part, and its characterization is available on the registry. We have tested the functionality of both its PETase component and its cellulose binding component. The part also includes an HlyA secretion sytem, but we were unable to confirm secretion.
Our hypothesis is that we can wrap bacterial cellulose around plastic to act as a scaffold on which the PETase can easily work in close proximity to the substrate. This should, in turn, increase the degradation rate. A model of our predictions can be found on the modeling page.

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