Team:Calgary/Results
Human Centered Design
IHP
Goal: In designing our project, our team wanted to make sure that our work was addressing real-life issues. We sought out the opinions of various stakeholders, including farmers, manufacturers, and leading scientists in our project design.
Result: We were able to integrate human practices at all 5 stages of our project design. First, we contacted farmers and oil producers in order to understand the scope of the green seed problem. Using our discussion with stakeholders as a starting point, our team began to ideate project ideas. In order to inform our design, we spoke to lead scientists to flag potential issues and improve our biological design. In order to evaluate our project, we brought our designs back to stakeholders to make sure our project aligned with their visions for the industry. Last but not least, we continued our conversation with various faculty members and the Canadian Grain Commission to further iterate on our design.
Future Directions: As we continue to expand our project, we hope to maintain our relationship with stakeholders at each level to make sure our project grows in the right direction.
Entrepreneurship
Goal: After identifying the green seed problem as a major problem within the green seed industry, our team looked into ways in which our solution could be implemented into the industry.
Result: Through our industry outreach, we were able to find that our emulsion system could be used to supplement clays in existing chlorophyll removal infrastructure to maximize chlorophyll removal and oil yield at a lower cost.
Future Directions: After the 2019 Giant Jamboree, we hope to patent our Emulsified Binding Protien (EBP) process, and move towards creating a start-up company for our design.
Education and Public Engagement
Goal: In order to strengthen the Albertan canola industry, our team aimed to use education and public engagement to enhance the skilled workforce, create proper infrastructure, and foster consumer acceptance of canola products.
Result: Through thorough education of new iGEM team members, discussion of synthetic biology principles with high-schoolers across the city, and engagement activities with the general public, we were able to address all three of these objectives.
Future Directions: We hope to expand our outreach efforts to reach even more people. We hope to continue educating future synthetic biologists through our synthetic biology education package.
Wet Lab
Goal: Our team aimed to design constructs and express and purify 6GIX, a water soluble chlorophyll-binding protein, for use in our emulsified binding protein (EBP) process.
Result: Our team successfully used Golden Gate assembly to create seven constructs to express the 6GIX protein. Six of these constructs contained each of the different signal peptides OmpA, MalE, TorA, YcbK, DsbA, and PhoA signal peptides. We also were able to create a construct without a signal peptide. Using the T7 promoter system and Ni-NTA chromatography, we were able to successfully induce and purify 6GIX. The correct protein size was visible at 21kDA in a western blot using Anti-His MAb (from mouse) and Anti-mouse IgG conjugated with HRP antibodies.
The 6GIX protein with PhoA, DsbA, and MalE signal peptides were successfully secreted into the periplasm of the cells, with the brightest protein band visible for secretion using the MalE signal peptide.
Future Directions: In order to further characterize the 6GIX protein, we hope to assess the binding affinity of 6GIX to chlorophyll.
Protein Emulsification
Goal: After obtaining a purified 6GIX protein sample, we aimed to provide a proof-of-concept for our Emulsified Binding Protein (EBP) system by showing that 6GIX is able to bind chlorophyll in an emulsion.
Result: Preliminary emulsion tests show that 6GIX is capable of binding chlorophyll in an emulsion. 6GIX is shown to have superior chlorophyll-binding capabilities to BSA.
Future Directions: We hope to induce and purify ModGIX, a 6GIX protein improved for stability, and compare its efficiency to 6GIX in emulsion.
Chlorophyll Repurposing
Goal: Our team aimed to characterise CBR, 7-HCAR, SGR, and PPH, which are four enzymes involved in the degradation pathway of chlorophyll to pheophorbide a.
Result: Using ICARUS, our universal spacer, we were successfully able to purify 7-HCAR and PPH using Ni-NTA chromatography. The correct protein sizes at 58kDa and 55.8kDa were visible in a western blot using Anti-His MAb (from mouse) and Anti-mouse IgG conjugated with HRP antibodies.
Through thin layer chromatography analysis of pheophytin, pheophorbide, and reactions involving our recombinant PPH protein, we were able to qualitatively show that pheophytinase is able to enzymatically convert pheophytin into pheophorbide.
Future Directions: In the future, we hope to purify and characterize the enzymatic activity of CBR and SGR, the two other enzymes involved in the chlorophyll degradation pathway.
Anti-Fungal
Goal: Our team sought out to determine the effect of pheophorbide a, a downstream of chlorophyll, on Sclerotinia sclerotium and Pestalotiopsis microspora
Result: We were able to show that pheophorbide a has an inhibitory effect on the mycelial growth rate of Sclerotinia sclerotiorum, but not on Pestalotiopsis microspora. This inhibitory effect was proven to be controlled by photo-activation and was positively correlated with increasing treatment concentrations.
Future Directions: We plan on further characterizing the effect of pheophorbide a on S. sclerotiorum through assessing the effect of daily reapplication of pheophorbide a. In addition, we would like to assess whether or not pheophorbide a could prevent S. sclerotiorum infection on whole canola plants.
Dry Lab
Following is an overview of the results gained in the Dry Lab for the 2019 iGEM Competition
ICARUS
1-2 paragraph overview of work done should link to pages and specific areas if mentioned
Emulsion Prediction
1-2 paragraph overview of work done should link to pages and specific areas if mentioned
Directed Protein Modification
1-2 paragraph overview of work done should link to pages and specific areas if mentioned
Emulsion Verification
1-2 paragraph overview of work done should link to pages and specific areas if mentioned
BOTs
The BioBrick Optimization Tool for synthesis was shown to be successful, as it could generate sequences with a lower idt score for the SGR sequence.
