Difference between revisions of "Team:OLS Canmore Canada/Results"

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<h1 class="title">The Design</h1>
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<h1 class="title">Introduction</h1>
 
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The project will use synthetic biology to create a novel fusion protein that can specifically bio-tag polyethylene terephthalate (PET) plastic, so that it can be sorted and recycled correctly. Synthetic biology is efficient, cost effective, and specific. The proteins, which are produced via a bacterial chassis called Bacillus subtilis, are created efficiently and at low cost. These proteins also provide high specificity due to a specific 3-dimensional shape that adheres selectively to PET polymers.  The 4 constructs that we have designed, with the help of our mentors and previous iGem teams, include:
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These are our list of results that we have successfully acquired through our two-year run developing this project. Special thanks to Lisa Oberding for helping with our Gene Design, Gene Synthesis, and Transformation.
 
</p>
 
</p>
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<br>
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<h1 class="subtitle">Gene Design<h1>
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<p>The 4 constructs that we have designed, with the help of our mentor Lisa Oberding and previous iGem teams (especially Tianjin 2016), include:</p>
 
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<ul class="standard">
 
<ul class="standard">
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<li>a polyethylene terephthalate hydrolase (PET-ase) fused to a red fluorescent protein, (or RFP) called mCherry, which give the protein its <b>colour</b> aspect. </li>
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<li>a polyethylene terephthalate hydrolase (PET-ase) fused to a red fluorescent protein, (or RFP) called mCherry, which gives the protein its colour aspect, </li>
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<li>a hydrophobin called BslA,</li>
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<li>a hydrophobin called BslA fused to mCherry, </li>
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<li>a PET-ase without the RFP, and </li>
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<li>a PET-ase without the RFP, and</li>
 
<li>a BslA without RFP.</li>
 
<li>a BslA without RFP.</li>
 
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</ul>
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<br>
 
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<p>
 
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A LipA secretion tag is added to each construct to signal the bacteria to secrete the proteins out of the cell for easier purification. We chose to use this Bacillus over E. coli because of its natural ability to produce hydrophobins, and because it is better at secreting proteins than other bacteria.  Bacillus is also naturally occurring in the environment, and has reduced risk for environmental contamination concerns.
 
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</p>
 
 
<br>
 
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<p>Special care was taken to ensure the sequences did not include any extra restriction enzyme sites and to design the coding regions to be expressed successfully in BOTH E. coli and B. subtilis cells.  See our parts pages for further details regarding these parts.</p>
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The PET-ase is an enzyme that naturally binds to PET plastic, and the mCherry RFP it is paired with will visually indicate when the protein has adhered. The hydrophobin is “water-fearing” and will therefore bind to several surfaces. However, for this project, it will be used to help adhere the PET-ase specifically to PET plastic. We are using the four proteins in combination with each other and test their effectiveness at tagging PET plastic.
 
 
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<table style="width: 40%; float: right;" >
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<table style="width: 40%; float: clear;" >
 
<tr><td><img  width="100%" src="https://static.igem.org/mediawiki/2018/d/d9/T--OLS_Canmore_Canada--prototypetext.svg"></td></tr>
 
<tr><td><img  width="100%" src="https://static.igem.org/mediawiki/2018/d/d9/T--OLS_Canmore_Canada--prototypetext.svg"></td></tr>
 
<tr><td class="imagecaptiontext">Implemented prototype, what could be seen in a sorting facility.</td></tr>
 
<tr><td class="imagecaptiontext">Implemented prototype, what could be seen in a sorting facility.</td></tr>
 
</table>
 
</table>
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<h1 class="subtitle">Gene Synthesis</h1>
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<p>
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The gene constructs we have created with the help of Lisa were synthesized by Bio Basic and shipped to us for transformation. The plasmids were ordered both in the standard pSB1C3 backbone, as well as a second “shuttle” backbone we hope to use in the future for testing on Bacillus specifically.
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</p>
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<h1 class="subtitle">Transformations</h1>
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<p>Our lab tried repeatedly to transform our cells.  We had success with E. coli but were unable to transform Bacillus subtilis on our own.  Thank you to Amino Labs who hosted us over the summer for a second (though unsuccessful) round of attempts at Bacillus transformation.  Thankfully our mentor, Lisa Oberding from Fredsense Technologies, was able to help us successfully transform both E.coli and B.subtilis cells with each of our four constructs.</p>
 
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<p>These pictures are our constructs in E.coli DH5alpha. The ones on the left are imaged under regular light and the picture on the right is under UV light. The coding system Lisa used for the test is:</p>
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<ulclass="standard">
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<li>E - PET-ase</li>
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<li>F - mCherry PET-ase</li>
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<li>G - BslA</li>
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<li>H - mCherry BslA</li>
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</ul>
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<h1 class="subtitle">Machine Prototype</h1>
 
<h1 class="subtitle">Machine Prototype</h1>

Revision as of 11:35, 17 October 2018

RESULTS

Introduction

These are our list of results that we have successfully acquired through our two-year run developing this project. Special thanks to Lisa Oberding for helping with our Gene Design, Gene Synthesis, and Transformation.


Gene Design

The 4 constructs that we have designed, with the help of our mentor Lisa Oberding and previous iGem teams (especially Tianjin 2016), include:


  • a polyethylene terephthalate hydrolase (PET-ase) fused to a red fluorescent protein, (or RFP) called mCherry, which gives the protein its colour aspect,
  • a hydrophobin called BslA fused to mCherry,
  • a PET-ase without the RFP, and
  • a BslA without RFP.


Special care was taken to ensure the sequences did not include any extra restriction enzyme sites and to design the coding regions to be expressed successfully in BOTH E. coli and B. subtilis cells. See our parts pages for further details regarding these parts.


Implemented prototype, what could be seen in a sorting facility.

Gene Synthesis

The gene constructs we have created with the help of Lisa were synthesized by Bio Basic and shipped to us for transformation. The plasmids were ordered both in the standard pSB1C3 backbone, as well as a second “shuttle” backbone we hope to use in the future for testing on Bacillus specifically.

Transformations

Our lab tried repeatedly to transform our cells. We had success with E. coli but were unable to transform Bacillus subtilis on our own. Thank you to Amino Labs who hosted us over the summer for a second (though unsuccessful) round of attempts at Bacillus transformation. Thankfully our mentor, Lisa Oberding from Fredsense Technologies, was able to help us successfully transform both E.coli and B.subtilis cells with each of our four constructs.


These pictures are our constructs in E.coli DH5alpha. The ones on the left are imaged under regular light and the picture on the right is under UV light. The coding system Lisa used for the test is:

  • E - PET-ase
  • F - mCherry PET-ase
  • G - BslA
  • H - mCherry BslA

    • Machine Prototype

    With our constructs design in place, we had to design a way of using them in a real life situation. Drawing on our experiences visiting real sorting facilities, and using the feedback and insights gained from the people working in this industry, we have designed a prototype using existing technology to adapt to our solution. A simplified description of our prototype includes the following steps:


    1. Incoming, unsorted plastics move along a conveyor belt and pass through a bath of our purified protein bio-tag.
    2. Our bio-tag selectively adheres only to PET plastics.
    3. Next all plastics will pass through a wash or rinse. The bio-tag is removed from any non-PET plastics.
    4. An optical scanner detects the fluorescent signature of mCherry on the PET plastics, and will separate it from the rest of the plastic.
    5. In future, similar bio-tags can be developed to selectively mark all other recyclable plastics using similar design principles.