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

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<h1 class="subtitle">The Subtitle</h1>
 
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<p>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. The project involves two proteins, a polyethylene terephthalate hydrolase (PETase) and a hydrophobin called BsIA, that is produced by a bacterium chassis called Bacillus subtilis. The PETase protein naturally binds to PET and would be paired with a red fluorescent protein called mCherry to visually indicate when the protein has adhered. The hydrophobin is “water-fearing”, therefore it will bind to anything, but for this project, it will help to adhere the PETase specifically to PET plastic. The project plan is to experiment with the use of both proteins, together and independently. If successful, the bio-tag would be proof of concept for a novel technology that can be implement easily in existing recycling facilities.
 
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Before incorporating it into the recycling facility, the protein would be isolated and purified, and the team will run numerous proof-of-concept assays. The next step in the project is prototyping. The team has explored a prototype which would use a streamlined linear process that involves both existing technology and new robotics to effectively sort plastics. Early business modelling suggests that this project is desirable by people, feasible with technology and viable as a business.
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<li>Preparation of competent B. Subtilis SCK6 cells for immediate transformation:</li>
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<li>2 days before transformation streak out a plate (1uL/mL final Erythromycin) of SCK 6 and grow at 37℃ overnight.</li>
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<li>Prepare seed culture: inoculate a single colony from the plate into 5 mL of LB-Erythromycin (1uL/mL) in a 50 mL Falcon tube. Incubate overnight at 37℃ at 200 rpm. </li>
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<li>Take the absorbency of the seed cultures using the spec. The OD600 should be approximately 1.0.</li>
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<li>Place 1000µL of overnight culture into a sterile Eppendorf tube for each transformation. Centrifuge and pour off the supernatant. Repeat 4 more times, to centrifuge the entire 5 mL of the overnight culture into 1 Eppendorf tube - pellet should be large. Do not discard the overnight falcon tube. </li>
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<li>Place 5 mL of Fresh LB broth (pre-warmed to 37℃) into the original overnight Falcon tube. Add 5 µL of erythromycin. Add 250 µL of 10% sterile xylose solution to the LB broth. Add 500 µL of this broth mixture to centrifuged cells in the Eppendorf tube (pipette up and down to resuspend cells). Transfer 500 µL of cells back into large Falcon tube. Incubate at 37℃ for 2 hours on the rotary shaking table.</li>
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<p>In summary, the OLS SynBio team is creating a novel protein bio-tag that will adhere selectively to PET plastics.  This product has the potential to revolutionize the recycling industry, and reduce the current practice of landfilling poorly sorted plastics. This will create a truly circular life cycle for plastic products.</p>
 
  
 
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Revision as of 04:09, 16 October 2018

EXPERIMENT

The Summary

In recent years, the issue of plastic pollution has become an overwhelming global crisis. Only 5% of all plastics are recycled and the rest ends up in landfills or oceans. When looking for a solution to this problem, the Design Thinking methodology learned at the Berkeley Program was applied. In the engagement with recycling stakeholders, the OLS SynBio team discovered that the issue is not the recycling of plastic, but instead the inefficient sorting of these plastics.


To further understand this issue, the team participated in many community outreach events. OLS Synbio consulted with Simon Robbins, the corporate manager of a local recycling plant, who provided guidance and insight of how the recycling cycle works. OLS SynBio also met with Peter Duck, the zero waste manager for the town of Canmore. Lastly, the team went to the Alberta Recycling Conference to learn more about how plastics are recycled in the community, and how big the team’s contribution would be. Stakeholder feedback helped to pivot and refine the project.

Some members of our team at the Canmore Sorting Facility.

The Subtitle

  1. Preparation of competent B. Subtilis SCK6 cells for immediate transformation:
  2. 2 days before transformation streak out a plate (1uL/mL final Erythromycin) of SCK 6 and grow at 37℃ overnight.
  3. Prepare seed culture: inoculate a single colony from the plate into 5 mL of LB-Erythromycin (1uL/mL) in a 50 mL Falcon tube. Incubate overnight at 37℃ at 200 rpm.
  4. Take the absorbency of the seed cultures using the spec. The OD600 should be approximately 1.0.
  5. Place 1000µL of overnight culture into a sterile Eppendorf tube for each transformation. Centrifuge and pour off the supernatant. Repeat 4 more times, to centrifuge the entire 5 mL of the overnight culture into 1 Eppendorf tube - pellet should be large. Do not discard the overnight falcon tube.
  6. Place 5 mL of Fresh LB broth (pre-warmed to 37℃) into the original overnight Falcon tube. Add 5 µL of erythromycin. Add 250 µL of 10% sterile xylose solution to the LB broth. Add 500 µL of this broth mixture to centrifuged cells in the Eppendorf tube (pipette up and down to resuspend cells). Transfer 500 µL of cells back into large Falcon tube. Incubate at 37℃ for 2 hours on the rotary shaking table.