Difference between revisions of "Team:UMaryland/Improve"

 
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<div class="subtitleText">Increasing the Efficacy of PETase</div>
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<h1>Improve</h1>
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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<h3>Gold Medal Criterion #2</h3>
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement.  Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
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The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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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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<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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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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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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