Team:Calgary/Results
Human Centered Design
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.
Goal: Since a main cause of the green seed problem is frost, farmers and the alberta canola association expressed how it could be useful to have better frost prediction.
Result: We designed software that can accurately predict long term daily-lows for temperature in a local town, Vulcan.
Future Directions: We want to increase accuracy, as well as increase its ability to predict frost using other parameters.
Goal: Farmers expressed displeasure with the current archaic method of grading seeds. We wanted to build an inexpensive method to standardize the grading of canola seed.
Result: We designed software, that when coupled with our lightbox to standardize conditions, could with a few tweaks very accurately grade canola seeds.
Future Directions: We want to increase accuracy and speed by changing our code to c++, as well as training a dataset with a convolutional neural net.
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.
Goal: In order to strengthen the Alberta 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.
Figure 1. SDS-PAGE gel showing whole cell lysate and Ni-NTA purification fractions for 6GIX without a signal peptide and an empty vector control. The marker used is the NEB colour protein standard. The arrow denotes correct band size of 21 kDa for the 6GIX protein.
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.
Figure 2. Western blot of whole cell lysate and Ni-NTA purification fractions for 6GIX without a signal peptide and an empty vector control. A His-tagged positive control was also included. The marker used is the NEB colour protein standard. The antibodies used were Mouse Anti-HIS-tag mAb (MBL) for primary antibody and Goat Anti-Mouse:HRP (Jackson ImmunoResearch Laboratories) for secondary antibody.
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.
Figure 3. SDS-PAGE gel showing periplasmic purification of 6GIX with six different signal peptides, no signal peptide, and an empty vector control. The marker used is the NEB colour protein standard. The arrow denotes correct band size of 21 kDa for the 6GIX protein.
Future Directions: In order to further characterize the 6GIX protein, we hope to assess the binding affinity of 6GIX to chlorophyll.
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.
Figure 4. Acid-activated clay, BSA positive control, 6GIX, and buffer negative control emulsions. BSA and 6GIX were emulsified at equal concentrations. The upper phase is a pure oil phase and the lower phase is a oil in water microemulsion. Processed and unprocessed canola oil samples were also imaged for reference.
Figure 5. Percent change in chlorophyll concentration in oil after emulsification with BSA and 6GIX.
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.
Goal: Our team aimed to characterise CBR, 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 HCAR and PPH using Ni-NTA chromatography. 10% SDS-PAGE gels were stained with Coomassie blue to reveal HCAR and PPH presence in the elution fractions of a Nickel-NTA column purification.
Figure 6. 10% SDS-PAGE HCAR and PPH Purification Confirmation. 10% SDS-PAGE was run at 100V for 15 minutes and then 180V for 35 minutes, then stained using Coomassie Blue. Lanes read left to right contain Color Prestained Protein Standard, Broad Range (11–245 kDa) as a ladder (NEB), PPH - post loading fraction, PPH - elution fraction 1, PPH elution fraction 2, HCAR - post loading fraction, HCAR - elution fraction 1, HCAR elution fraction 2, pSB1A3 (plasmid control in BL21) - elution fraction 1, and pSB1A3 (plasmid control in BL21) - elution fraction 2. NEB Ladder is shown on the left. HCAR is 58 kDa and PPH is 55.8kDa. This SDS-PAGE Confirmation is replicate 4/5, other replicate images are available here.
The identity of HCAR was confirmed in a western blot using Anti-His MAb (from mouse) and Anti-mouse IgG conjugated with HRP antibodies.
Figure 7. Western Blot HCAR Purification Confirmation. 10% SDS-PAGE was run and transferred to a PVDF membrane. The antibodies used were Mouse Anti-HIS-tag mAb (MBL) for primary antibody and Goat Anti-Mouse:HRP (Jackson ImmunoResearch Laboratories) for secondary antibody. ECL was used to visualise. Lanes from left to right on the SDS-PAGE were ladder (Color Prestained Protein Standard, Broad Range (11–245 kDa) (NEB)), ~60 kDa protein (positive control) in two lanes, pSB1A3 (plasmid control in BL21) - elution fraction 3, HCAR - elution fraction 1, HCAR - elution fraction 2, HCAR - elution fraction 3. HCAR is 58 kDa.
Through thin layer chromatography analysis of pheophytin, pheophorbide, and reactions involving our recombinant PPH protein, we were able to qualitatively show that pheophytinase (PPH) is able to enzymatically convert pheophytin into pheophorbide.
Figure 8. Thin Layer Chromatography Analysis of Pheophytinase Reactions. Samples were eluted using a methanol:hexane (70:30) solvent system on a silica plate. Lanes (read left to right) contain 24 hour reactions using Nickel-NTA purified PPH elution fraction 3, 2, 1, whole cell lysate (containing recombinant PPH) fraction 2, 1, pheophytin + reaction buffer solution, pheophytin + pheophorbide a + reaction buffer. Reaction buffer: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100. This is one of three replicates. The left (8A) figure is the raw image taken using an LG G6 Camera whereas the right (8B) is the same image with modified brightness:contrast ratio using the imaging software FIJI.
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.
Goal: Our team sought to determine the effect of pheophorbide a, a catabolite 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.
Figure 9. Mycelial growth of Sclerotinia sclerotiorum with pheophorbide a in dark (6A-left) and in light (6B-right) conditions. Five treatments were applied as treatment discs impregnated with pheophorbide a, solubilized in 25% acetone (0, 5, 15, 25, 35 mg/mL). The 0 mg/mL treatment was 25% acetone. Two treatment discs for each concentration were placed 1.5 cm from the epicentre of the fungal culture. Growth was tracked for four days after culturing. Measurements were taken once a day from the epicentre of the original culture to the edge of the mycelial growth toward the disc. Maximum growth is recorded at 4.2 cm due to the potato dextrose agar plate capacity. Light conditions were conducted using 1400 lumen white LED light at a distance of 25 cm from the plate. Data points are summarized in Anti-Fungal Table 6A and 6B here.
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
Goal: Our team aimed to create a “universal spacer” that would mitigate unwanted electrostatic interactions between proteins and their tags.
Result: We were able to design ICARUS, which we used on 7-HCAR and PPH. We were successfully able to purify both of these proteins, which had not been done before in literature.
Future Directions: We hope to character the efficacy of this spacer with respect to other commonly used spacers in synthetic biology. This would allow for synthetic biologists worldwide to have more choice in designing parts that work for their specific protein and tag.
Goal: Our goal was to create a model to predict the Winsor type of an emulsion made with different concentrations of aqueous phase, oil phase, and surfactants.
Result: We were able to generate ternary phase diagrams that informed five different emulsion conditions tested in the lab. Using these emulsion conditions, we were able to optimize our 6GIX emulsion.
Future directions: We hope to use these models to optimize the emulsion of ModGIX, our improved 6GIX protein.
Goal: Our team aimed to create an improved 6GIX with even greater stability in a bi-continuous emulsion system.
Result: Our team was able to use genetic modification algorithms to produce ModGIX, an improved 6GIX protein with enhanced stability. The stability of this protein has been characterised in silico.
Future Directions: We would like to induce and purify ModGIX, and compare its efficiency in chlorophyll capture to 6GIX.
Goal: Our goal was to predict how 6GIX, our water soluble chlorophyll-binding protein, would behave in an emulsion.
Result: Protein dynamics models show that 6GIX is stable in an aqueous solvent, denatures in a non-polar solvent, and forms a stable tetramer in emulsion.
Future Directions: We would like to assess larger-scale interactions in our system, such as multiple emulsion bubbles over a longer timespan. In addition, we would also like to run chlorophyll binding models to assess the binding ability of 6GIX and ModGIX.
Goal: We sought to develop a tool that would be able to simplify the genetic construct generating process for hard-to-synthesize sequences.
Result: With BOTs' advanced SPEA2 algorithm, we were able to optimize sequences for both expression and ease-of-synthesis. Using this tool, we were able to reduce IDT Gene Fragment Synthesis scores from over a hundred to 7.
Future Directions: We would like to deploy this software onto the website we have designed, to allow other iGEM teams and synthetic biologists to use our technology with greater ease.
Goal:Recognizing that protein modification is a discipline that many iGEM teams struggle with, we sought to create a tool to simplify the process of making informed changes to their amino acid sequences.
Result: We created iGAM, an R package, which we used to successfully generate our modified 6GIX protein, ModGIX.
Future Directions: We hope to integrate the workflow of iGAM with pathway enzymes, and create a software tool for further accessibility.
