Providing a solution to the green seed problem could be the key to alleviating losses felt by those in the canola industry. Our teams’ vision was to make our project that solution. In order to do so, we had to ensure that we created something that was effective, usable, and targeted areas in which people were impacted the most. The only way in which to achieve this goal, was to integrate our end-users in creating our solutions.

We utilized a human-centered design process and identified a set of steps to guide our work. This lead to integration with key stake holders throughout the project lifecycle, and ultimately the all-encompassing solutions found in yOIL.

Before BioBrick design and lab work began, there were questions we needed answered. We needed to know what exactly the green seed issue entails, how large it is, who is impacted, and what is being done to stop it.

To answer these questions, we identified four groups of people that we needed to speak with. Our primary research indicated that there are two main entities impacted by the green seed issue: farmers and oil producers. In addition to these two, we also identified agronomists and organizational bodies that worked within the canola oil industry. From here, we initiated conversations about the green seed issue and discussed how we could utilize synthetic biology as a solution.

Craig Shand: The impact on the farmers...

Craig Shand is a local Southern Albertan Farmer and Agronomist. It was essential for us to understand the exact effect green seed has on Canadian agriculture from someone directly in the industry experiencing the losses.

The green seed problem creates losses at the canola crushers, and those losses are passed on to farmers like Craig. When farmers are already operating on a sliver of a profit margin, the downgrade of crops from $10 per bushel to $5-6 per bushel can make or break a growing season. Or as Craig succinctly puts it: “Green is the difference between profit” ,

Our project needed to strive to keep money in the pockets of farmers. Talking to Craig informed our team of how we could potentially leverage our scientific advances to help Albertan farmers. Our solution would have to go beyond simply purifying chlorophyll from oil. We would need to create a market for green canola seed.

As an Agronomist, Craig is heavily involved in field scouting and trying to help as many farmers as possible manage green seed. He identified two major problems: the first being that the Albertan growing season is relatively short and is dictated by the frost-free time period. Canola seeds must mature before frost hits, otherwise green seeds will form. Frost is an ephemeral boogeyman to farmers, you do not know when it is coming until it is too late.

Secondly, when farmers take their harvest to grain elevators for grading, that grading process is done manually. It is inefficient, time consuming and worse, highly subjective. Craig highlighted the fact that you could take your harvest to two different grain elevators and get two different gradings. Gradings being the difference between selling at a premium or selling at a loss, Craig opened our eyes to a problem that can be addressed.

Ward Toma: Green seed defines a growing season...

Ward Toma is the General Manager of the Alberta Canola Producers Commission (ACPC),an organization which supports the “long-term success of canola farmers in Alberta through research, extension, consumer engagement, and advocacy for canola farmers”(https://albertacanola.com/about/). As General Manager, Ward is responsible for overseeing the ACPC and is often the public face advocating for Albertan canola. His background in Agricultural Economics was a perfect jumping point into a long, detailed conversation about where Albertan agriculture needs help.

Starting with Green Seed, Ward confirmed what we started to fear after talking to Craig: the success of a farmer’s growing season is largely defined by green seed content. By his account, 6-7% of an annual crop consists of green seed that is blended into together into the bulk of the crop, reducing the value of the crop. He suggested that potentially recovering the chlorophyll and making it into something valuable would be unique and profitable in comparison to the current clay method. Creating a market for green seed and increasing demand would give leverage to the farmer when negotiating prices at the grain elevators. Ward confirmed that green seed is the main factor affecting canola crop value throughout a harvest cycle.

Ward was also kind enough to give us a lead on another excellent resource, pointing us towards Dr. Veronique Barthet, a researcher at the Canadian Grain Commission.

Dr. Veronique Barthet: The pitfalls of processing...

Dr. Veronique Barthet is a Research Scientist and Program Manager for Oilseeds Monitoring and Research at the Canadian Grain Commission’s Grain Research Laboratory. Her areas of expertise lie in researching grain quality factors, seed oxidative stability and developing analytical quality measurement methods. She was specifically able to elucidate how the Grain Research Laboratory quantifies the quality of crops, such as canola, in an objective manner.

From Dr. Barthet, we learned that the Canadian Grain Commission relies mostly on spectroscopy and high performance liquid chromatography (HPLC) to determine the chlorophyll content of green seeds. She also told us that chlorophyll a is in the highest abundance in green seed oil, a consideration which would affect our project design

Dr. Barthet’s knowledge of the current method for purifying chlorophyll from canola oil, the acid-activated clay method, also proved to be invaluable to our project development. According to her, the clay used in the purification process is disposed following use, highlighting that creating a re-usable method would bring economic value to the oil processors. The acid-activated clay method is the only purification method that she is aware of meaning that is the only technique we would need to outperform.

Finally, Dr. Barthet emphasized that any solution we propose would have to be integrated into current oil processing plants, and would ultimately need to be commercially and economically viable. She believes that as long it is commercially viable, an alternative to bleached clay would be welcome in the canola oil processing community.

James Rea: A tour of the oil processing steps...

Look at email JD sent

I CAN'T FIND AN IMAGE OF HIM :(

Pleasant Valley Oil Mills: Something something...

How pleasant

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Autumn Barnes: What can farmers do farmers do now...

Autumn Barnes is an agronomist with the Canola Council of Canada

Angela Brackenreed: The canola oil industry is a pipeline...

Angela Brackenreed is an Agronomy Specialist with the Canola Council of Canada. We spoke with Angela to assess the viability of pursuing a project handling the rampant green seed problem. Since we spoke to her at the relative onset of our project, we valued her ability to give us a generalized outlook on the canola industry and give us an idea of the movers and shakers in the bustling Canadian Canola industry.

We learned that farmers in particular can use a few agronomic techniques to mitigate the impact of green seed on their crops. If a farmer can get an idea of when frost will occur, they can then accommodate the weather when choosing when to seed and when to swath. However, Angela made us realize that the industry follows a production pipeline of canola oil, from farmer to family tables, and the green seed problem effects every person along the way. We were surprised to learn from Angela that the canola industry is very accepting of genetically modified organisms, and we would just need to manage public perception if we designed such an approach. However, it was relieving to learn that 100% of canola grown is genetically modified and that the industry itself would not have ethical qualms to integrate a synthetic biology solution.

Through our discussions with these industry members, we learned that every stage of canola oil production faces tremendous losses due to the green seed problem. With a consumer market desiring pure oil, the green seed problem forces the players in the production pipeline to somehow deal with the green oil produced. However, farmers like Craig have no tools to plan for unexpected frost or drought, and gamble their harvest every year on unpredictable whether. As Angela and Ward confirmed, this leads to the farmers inevitably producing green seeds, which get graded as low quality and are sold at a lower price. Furthermore, we learned from Craig that the process to grade green seeds is not standardized or objective, making the system less fair. Additionally, the oil processors are forced to spend more time and money to purify the oil produced from the green seeds they receive, but Dr. Barthet stressed the unsustainability of the archaic acid-activated clay purification method, as it is not reusable, environmentally friendly, and causes loss of the oil itself. Every person we talked to asserted the impact green seed has had on them and emphasized the need for new solutions that would mitigate the losses incurred.

So what can we do to help this industry?

Focusing on the issues stressed in our discussions, we started brainstorming various dry lab and wet lab solutions that would solve the problems caused by green seed.

The clay method is not reusable, environmentally friendly, or selective to chlorophyll, thus, we needed to come up with a solution that tackled these issues. Proteins are organic and are made to be highly specific to their substrate, so we thought to design a system that utilized a protein to capture chlorophyll, but we weren’t confident as to which chlorophyll-binding protein to use, how it could be used in an oil environment, and what other considerations we needed to be aware of.

To answer these questions, we spoke to protein bio-chemists, micro-biologists, plant biologists, and chemical engineers who gave us insight into how to design our system.

Dr. Gordon Chua: Choosing the right chassis...

We spoke to Dr. Gordon Chua, a professor in the biological sciences department at the University of Calgary , about our initial project design. Dr Chua’s research interests focus on integrative cell biology.

At this point in the project, our team had just started literature review on chlorophyll-binding proteins, and we did not know much about their structure, function, or how they could be expressed. Our team had only looked at using bacteria as a chassis for our system, but Dr. Chua suggested we also consider yeast cells as a possible chassis. If we choose a eukaryotic protein, there is a chance it would be better expressed in yeast, a eukaryotic chassis. He also suggested that we consider a variety of chlorophyll binding proteins for use rather than just one.

Using this advice, our team investigated a variety of different chlorophyll binding proteins of two major families: membrane-bound proteins and water-soluble proteins. After thorough literature review, our team settled on using a water-soluble chlorophyll binding protein derived from L.virginicum, a plant in the mustard family. Although this protein was derived from a eukaryotic organism, previous literature showing successful expression of this protein in bacteria led us to our choice of E. coli as a chassis.

Dr. Joe Harrison: Membrane-bound proteins and biofilms may not be the best approach...

While investigating different types of chlorophyll-binding proteins, our team entertained the idea of using a biofilm to express a membrane-bound chlorophyll binding protein. This would require filtering oil through the biofilm to remove chlorophyll from the oil. Our team contacted Dr. Harrison, a microbiology professor who studies biofilms at the University of Calgary to gain insight on how they could work within our system. Dr. Harrison advised us to consider the amount of chlorophyll present in the oil compared to the amount of protein that can be present on the surface of a biofilm layer. He was not sure if we would be able to produce enough protein on the surface of a biofilm to capture all of the chlorophyll present.

Dr. Harrison also advised us to consider the consequences of having bacteria in direct contact with the oil being processed. In addition to food safety concerns, it is possible for the bacteria to metabolize the oil it is in contact with. However, Dr. Harrison also suggested that the oil metabolization could be mitigated by killing the bacteria using UV light or autoclaving, which would not alter the structure of the protein. However, this would mean that the protein needs to be fully folded before exposure and binding to chlorophyll. After further literature review of membrane-bound chlorophyll binding proteins, we determined that the protein would have to fold in the presence of chlorophyll for it to properly bind; thus, our team decided to move away from the use of biofilms and membrane-bound chlorophyll binding proteins.

Dr. Marcus Samuel: What research is being done...

We spoke to Dr. Raymond Turner, a professor in the biochemistry department at the University of Calgary, to discuss our emulsified binding protein (EBP) system.

Dr. Marcus Samuel is a plant biology professor at the University of Calgary, who has worked on further understanding the de-greening process in canola seeds. Our team spoke to Dr. Samuel to understand what research is being done to address the green seed problem. Dr. Samuel identified the green seed problem as the largest economic problem in the canola industry, and emphasized the need to create a better solution than the current acid-activated clay purification method. He told us that the acid-activated clay purification method can be problematic in expenses and disposal. He believes that our solution is novel for its synthetic biology application, which stands out amongst the largely chemical processing methods that are in use.

Dr. Samuel also gave us some pointers about our project design. He suggested that we model our chlorophyll binding protein before production to observe its stability and binding ability before we commit to protein production and purification, which led to our team’s protein dynamics models. He also advised that we consider the need to scale up the process for an industrial application. Keeping this advice in mind, our team explored multiple options for inducible systems to use for protein production. For our proof of concept in the laboratory, we decided to use the IPTG-inducible T7 system. However, our team plans to experiment with a xylose-inducible system, which is less expensive for industrial use, moving forth.

Dr. Ian Lewis: How to utilize a water-soluble protein in a hydrophobic environment...

We spoke to Dr. Lewis of the biochemistry department at the University of Calgary about our plan of using a water-soluble chlorophyll-binding protein (WSCBP) to purify chlorophyll from canola oil. We approached him for his expertise in protein dynamics; we wanted to see if he would be able to flag any issues with our use of the WSCBP. Dr. Lewis was enthusiastic about how this idea would allow us to increase the amount of protein relative to the chlorophyll in the oil.

Dr. Lewis advised that we consider the industrial scalability in the production of our protein. However, he also brought up that our water-soluble chlorophyll-binding protein has a hydrophilic exterior, and thus would not function in a hydrophobic environment such as oil. He explained that we would need to keep the exterior of the WSCBP hydrophilic - otherwise, our protein would denature immediately. With this advice in mind, our team sought out to find a way to keep our hydrophilic WSCBP in aqueous solution while still allowing it to come into contact with chlorophyll in a hydrophobic environment. This led to the idea of an emulsion system, which maximises the surface area between the aqueous and oil phases whilst keeping the WSCBP intact. Furthermore, by emulsifying our protein we would prevent it or the bacteria from being in direct contact with the oil, addressing a prior concerns about food safety.

Dr. Raymond Turner: Proteins need to be purified before emulsification...

We spoke to Dr. Raymond Turner, a professor in the biochemistry department at the University of Calgary, to discuss our emulsified binding protein (EBP) system.

Due to his expertise in protein stability and folding, we asked if there were any immediate areas of concern for him regarding our EBP system. Dr. Turner was optimistic about the function of our protein in an emulsion, but he stressed the need for purified protein to be used in our emulsion system to ensure that bacterial products would not be in the final product. Along the same vein, he emphasized that every material used in the processing of the oil has a chance of making its way into the final product, and thus we need to be careful about the materials that we use in processing. For example, if we use a nickel-NTA column to His-tag purify proteins, the final product could contain traces of nickel, which would be a problem for those with nickel allergies.

Realizing our time and resource limitations, Dr. Turner suggested that a proof of concept could involve a His-tagged protein binding to a nickel column, but the tag would need to be changed for an industrially scaled system. Following his advice, our team decided that for our lab work this summer, the use of a His-tag would be ideal. However, we performed a literature review of other tags that could be used in its place in an industrial system, such as a galactose-binding protein tag or biotin-carboxy carrier protein. Additionally, he suggested that secreted the protein could also minimize the costs of purification, and that trying different signal peptides with our protein could be a consideration.

Dr. Randall Weselake: Proteins should be reusable...

We spoke to Dr. Randall Weselake, a retired plant biochemist and former professor of biochemistry at the University of Alberta. His research involved the study of oil formation in seeds like canola, flax, and safflower, alongside ways to encourage plants to produce more oil for use in foods and the industry. Due to his expertise in plant oils and the canola industry, we wanted to discuss the value of our emulsified binding protein (EBP) system, and address any potential issues we would face in its execution.

Dr. Weselake heavily emphasized the value of cost in the industry. He thought a lot of the value of the project came from whether or not our protein was reusable. Upon validating binding, he advised that we investigate ways to remove chlorophyll from the binding protein so the protein can be reused. He also emphasized the importance of system efficiency; the more our system optimizes binding and reducing product loss, the better. This motivated our investigation of phase diagrams assessing different aqueous, oil, and surfactant concentrations in our emulsion to maximize our oil yield without compromising binding ability of our protein.

Dr. Marie Elizabeth Fraser: Movement of the chlorophyll molecules in the emulsion...

Our team had the opportunity to speak with Dr. Fraser, a biochemistry professor at the University of Calgary. She provided us with valuable advice regarding the proof of concept of our emulsion system, and the general feasibility of our chlorophyll extraction method. Her main concern was whether the chlorophyll would be able to cross the oil-water interphase and actually contact the chlorophyll binding proteins. As a future direction, she suggested that we look at the properties of lipases and see if we could any to make our protein more stable.

Additionally, she emphasized the importance of creating an emulsion with high stability. We took this advice to heart and created a number of models to ensure an emulsion of high stability would be made for our chlorophyll extraction process. Dr. Fraser also remarked that one of the issues in our process would be the industrial scale-up of our proof of concept due to the large quantities of oil processed in a plant. She also suggested that secretion of protein would be more ideal as it would lead to less chance of protein degradation compared to the extraction of protein from the cytoplasm.

Dr. Michael Kallos: Designing an efficient emulsion system...

Dr. Kallos...

Chlorophyll cannot be removed from the acid-activated clay chlorophyll after it is bound; however, by using chlorophyll binding proteins the chlorophyll can be released after. In our initial discussions with Ward Toma, he suggested that repurposing chlorophyll could bring new revenue into the canola industry and mitigate some of the losses caused by green seed. Dallas Gade further confirmed that valuable byproducts could be useful to offset expensive input costs. Pheophorbide a is a natural catabolite of chlorophyll that has been investigated recently for its photosensitizing abilities in experimental anti-cancer and anti-fungal treatments. Seeing how lucrative the product is, we decided to genetically engineer part of the chlorophyll degradation pathway in E. coli to produce pheophorbide a.

Producing functional eukaryotic enzymes in bacteria can be a challenge, especially those involved in a specific pathway. We decided to talk to a microbial biochemistry and a plant biotechnologist about the feasibility of our idea.

Dr. Raymond Turner: Designing in vitro enzymatic degradation pathways...

In addition to providing advice on our chlorophyll extraction system, Dr. Turner also had a few suggestions for designing our genetically engineered chlorophyll degradation pathway. He confirmed that producing each protein as its on individual construct may be good for laboratory proof-of-concept, but also suggested to look at linking the proteins together to create one conglomerate protein with each of the enzymes individually functioning, as it would increase the efficiency of the degradation pathway. Furthermore, he confirmed that creating an external pathway with connected compartments for each enzyme and its cofactors, would be feasible and has been done before.

Dr. Gijs Rooijen: Thinking about industrial integration...

Dr. Gijs van Rooijen is the current Chief Scientific Officer of Genome Alberta. He obtained his MSc in Molecular Sciences from the Agricultural University in Wageningen and a PhD in Plant Molecular Biology from the University of Calgary. Dr. Gijs worked for SemBioSys Genetics and (co)authored and co(invented) 25 issued patents in the US.

The goal of the meeting was to obtain a professional opinion regarding the value and feasibility of the project, and the issues we would face in terms of its execution. We also wanted to talk more about enzyme essays for our specific proteins.

Dr. Gijs expressed concerns about scaling up our project idea, especially since we are using proteins and purifying them with His tags which would be very expensive in the long run. He had some reservations with binding affinity to chlorophyll and yielding enough CBP from E.coli to make it worthwhile. Additionally, loss of product at each step would be inevitable. He stated that 10% of the work is the proof of concept but 90% is the optimization. In terms of biotechnology, a project would have more value if the technology is something “people are screaming for”. We should also consider whether the stakeholders are looking for innovation or whether they are happy with the status quo. To make a successful startup, “Helping farmers is a noble goal” but at the end of the day you need to deal with people looking at spreadsheets. Dr. Gijs suggested testing pheophorbide on breast cancer cell lines, or other potential applications to increase the value of the project. Being a plant biotechnologist he thinks that genetic modification at the crop level would be the best solution because it is best to tackle a problem at the base level. Ultimately, Dr. Gijs thought we would face many hurdles to develop the project into a commercial system.

Craig, Angela, and Ward, stressed two major hurdles farmers face when they produce and sell their seeds to oil processors; the unpredictable weather and the unstandardized seed grading system. We turned to computation as a solution to address these problems. Current weather forecasts cannot predict weather 3-4 months into the future, preventing farmers from employing agronomic techniques that could save their crops. Therefore, we wanted to see if we could design a long-term weather prediction system for farmers to use.

The current grading system involves a worker manually crushing a batch of 500 seeds and identifying each seed's colour based on a colour chip. The percentage of green seeds found denotes the grade of the batch, with a lower grade indicating higher green seed content. This process is very subjective, as it relies on the human eye to determine colour, and therefore grade. Farmers need a standardized system that will produce the same result every time, so they do not incur loss due to human subjectivity. We sought expert advice to build a machine that can solve this problem.

Jeff Danielson: What is the current grading system like...

1. Do you believe there is value to standardize % Distinctly Green Seed with a tool?

Yes, a more uniform result that removes the variability of individuals perception of color would be beneficial.

2. How is % Distinctly Green Seed currently evaluated at your elevator?

The process is done by counting 500 seeds with a canola “ruler”, then the seeds are transferred to a piece of masking tape, the seeds are crushed flat using a hand roller. Seeds are crushed flat in order to expose the inside of the seed. The seeds that qualify as distinctly green (DGR) are then counted and a percentage is calculated. The Canadian Grain Commission provides a visual color reference guide to help determine what is distinctly green and what is not distinctly green. This whole process can take anywhere from 30 seconds to a couple of minutes depending on the graders ability and the canola quality.

3. Do you know how % Distinctly Green Seed gets converted into a dollar amount?

The Canadian Grain Commission dictates the allowable tolerance for distinctly green in all three grades of Canola: Canola 1 Canada can have a maximum of 2% DGR, Canola 2 Canada can have a maximum of 6% DGR, Canola 3 Canada can have a maximum of 20% DGR. The price for these different grades or qualities of Canola is set by standard market forces of supply and demand. The problem with DGR is that it takes more time and resources to process and purify the finished oil. So this time and resources means increased expenses which means canola with DGR is less valuable. Canola crushing facilities would know the actual cost to process higher levels of DGR.

4. Who do you think will use a tool to standardize % Distinctly Green Seed?

Primary elevators, port terminals, crushing facilities, Canadian Grain Commission, independent service providers, some producers

5. What is the frequency at which suppliers and yourselves disagree on the grade of the canola?

The tolerance for DGR is quite wide within each grade, so there tends to be limited disagreement. The variability of the human eye and interpretation of color, can lead to disagreements around samples that are on the margins of the grade tolerances. Keep in mind that DGR canola is quite uncommon. Canola is a very hardy plant and tends to reach full maturity in most crop years. There can be years where there is essentially no DGR canola in the harvest.

6. Would you rather use a physical device or an app to standardize Canola Grading?

As long as it works consistently, is accurate and easy to use it wouldn’t matter.

7. Do you know anyone that has access to images of counts/quality of canola seeds? We can use this information to accurately build our device.

The Canadian Grain Commission would be able to provide you with the Canola Color Reference material. There is a GCG office located in Calgary. I can provide you with samples of green canola and referrals of where to buy supplies for crushing canola for grading.

Dr. Usman Alim: Analyzing the colour...

The team talked with Dr. Alim to gain some more context on certain aspects of the seed grading project. Namely, we wished to learn about methods of analyzing digital colour information and relating it to cut-off points based on a colour chip. This is due to the fact that current seed grading is done by comparing seed smears to a physical colour chip. As Dr. Alim’s areas of interest include applications of signal processing to computer graphics and visualization, he was quite helpful in pointing us in the right direction.

Dr. Alim initially brought up deep learning approaches like with convolutional neural networks, but the team explained that a lack of labelled seed data precluded this methodology from being used. As an alternative, Dr. Alim suggested using clustering methodologies to identify pixel clusters for yellow seeds and distinctly green seeds, even if arbitrary cut-off surfaces needed to be applied. These clusters would utilize different colour spaces, as colour spaces are essentially data transforms. Additionally, Dr. Alim brought up the Lab colourspace, as it designed so that even small differences in values correspond with human-visible differences. Unfortunately, when exploring the colour spaces of the seeds, there was too much data and too much overlap in pixel values between seeds for clustering methods to be applied. However, examination of different colour spaces revealed that they could be represented as geometric volumes, leading to the current approach of using colour space distance calculations for pixel analysis.

Sean Mason: Building the hardware...

Sean Mason was a mentor for the hardware of a 3rd year mechanical engineering student at UofC with years of experience as a machinist, was critical in concretizing mean green machine. Our team had no previous experience with DefinitionCADs or working with machine shops. Sean Mason acted as a mentor for designing parts in Solidworks, design considerations, measurements, tolerances, who to talk to for sourcing the parts, and choosing the right materials for the job. Whilst it remained an iGEM project, Sean Mason stood right behind us and made sure we were doing things correctly in Solidworks by providing tutorials on different aspects of Solidworks and more than once made us consider significant portions of the design to add functionality or ease of use. Ideas such as Definitionchamfers, which were never considered were added in due to his expertise in creating real-life projects that work. He also oversaw safety for drilling, sanding, and glueing for the first assembly of the machine. His ideas on how to create an easy-to-assemble standardization tool helped us create the way it is now.

initial contacts into the

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CanaolaPALOOZA The agronomy event of the summer...

Once we designed and established our various dry and wet lab projects entailing yOIL we attended CanolaPALOOZA. An annual event held in Lacombe, Alberta where canola experts all around Western Canada come together to discuss the issues facing the industry and the new methods to combat them. Many of our initial contacts including Ward Toma and Dr. Veronique Barthet were also in attendance. This event was an opportunity for us to propose our ideas to everyone in the industry and get their feedback on the commercial viability of our project.

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Visiting Richardson Oilseed

We visited Richardson

Stuff stuff

4. Evaluate

Pheophorbide- A unique solution to an industry problem

Attending CanolaPALOOZA allowed us to evaluate our project to see if we were effectively accomplishing our goal of helping the canola industry. Overall, we got very positive feedback on our projects, but we did learn about other problems the industry faces aside from green seed. Canola suffers from a fungal disease caused by Sclerotinia sclerotiorum leading to huge losses in crop yield annually (REFERENCE???) . This was interesting to us because our product, pheophorbide, is an anti-fungal agent that could be used to address this problem.

We decided to further investigate the effect S. sclerotiorum has on the canola industry (particularly on the farmers) and how we could use pheophorbide as a unique solution to combat it.

Dr. Ronald Moore: Photodynamic therapy and its uses...

Our early background research identified pheophorbide as a candidate for use as a therapeutic agent for photodynamic therapy. Photodynamic therapy (PDT) is a medical treatment which utilizes photosensitizing compounds, such as Pheophorbide, in the presence of specific wavelengths of light in order to produce a directed cytotoxic effect. PDT is currently being investigated for applications in cancer treatment and there already exist several commercial photosensitizers such as Photofirin. We were interested in Pheophorbide’s potential as a PDT photosensitizing agent, but we had to talk to someone to learn more about it.

Dr. Ronald Moore is a professor of Surgery and Oncology at the University of Alberta and currently serves as the Mr. Lube Chair in Uro-Oncology Research. He has over 25 years of experience in researching novel therapies for genitourinary malignancies.

Our discussion with Dr. Moore gave us an excellent overview of PDT’s current usage as well as what considerations doctors make when choosing a photosensitizing agent for PDT. Generally, the world of experimental cancer research is constantly in flux, changing with new developments and discoveries. While the current consensus is positive, tumorous cells substantially retain photosensitizers, the future's uncertain and the demand for photosensitizing agents in the future is shrouded in darkness.

Following our conversation with Dr. Moore, it was immediately apparent that we could not put all our eggs into one PDT-basket. We needed to explore other ways. During our literature review, we identified PDT as a potential method to treat fungal infestations. After our meeting with Dr. Moore we realized that pheophorbide has more potential as an anti-fungal agent than as an experimental cancer treatment drug.

Dr. Heather Addy: Designing effective fungus experiments...

After the meeting with Dr. Moore, we immediately had to learn more about working with fungi and anti-fungal assays. Through background research, we had the idea to use the “drop method”, applying drops of pheophorbide to different locations on an agar plate, with varying concentrations to see if it inhibited fungal growth. However, we needed to validate our experimental design. So we met with Dr. Heather Addy.

Dr. Heather Addy is a mycologist and plant biologist who specializes in plant-fungal interactions at the University of Calgary. Her expertise in the field meant we absolutely had to talk to her to inform the design of our anti-fungal assays.

Dr. Addy gave us the idea to use the “disc method”, immersing paper discs in solubilized pheophorbide, placing them around a fungal culture placed in the centre of a potato dextrose agar plate. The fungal colony would grow and eventually come into contact with the pheophorbide discs. Over the course of the experiment, we would measure the distance from the centre of the plate to the edge of the fungal colony’s growth. If pheophorbide does in fact have an inhibitory effect on fungal growth, then there would be a decreased rate of growth for the portions of the colony interacting with pheophorbide.

Dr. Addy generously put us in touch with Fran Cusack, a Biological Sciences Technician who prepares fungal samples for classes. Fran was kind enough to provide us with samples of Pestalotiopsis microspora and S. sclerotiorum, the same fungus which commonly afflicts canola crops.

We now had the information and tools at our disposal, to begin testing pheophorbide’s application as an anti-fungal agent. However, there was still a gap in our knowledge regarding the what the average Albertan farmer goes through when faced with fungi, like S. sclerotiorum

John Mayko: Fungal treatments must be prevantative...

To say John Mayko is merely an Agronomy Specialist from Mundare, Northern Alberta would vastly undersell his involvement with the Alberta Canola industry and his contribution to yOIL.

Receiving his Masters degree in Engineering Agrology from the University of Alberta, John is the president of Apex Agrology Services and currently sits on the Board of Directors for the Alberta Canola Producers Commission. His many years as a Senior Agri-Coach meant he could give us a clear indication of how fungi affect Albertan farmers.

John himself has had to deal with fungus. According to him, “Anybody growing canola in Alberta will have to deal with it”. There is no question that fungus is an issue for canola farmers, but what is being done about it?

Unfortunately, there is no fix for fungus. Once a crop has been afflicted by fungal blight, it must be discarded, there is no turning back the clock. At the early bloom stage, every farmer must make a decision whether or not to apply anti-fungal treatments to their crop. It is a costly proposition ($20-$30 per acre according to Dr. Kelly Turkington) which does not give a 100% guarantee.

From John, we learnt that our pheophorbide application would have to be preventative not prescriptive. He also gave us the indication that for our product to be viable, it would have to be cheaper to apply than current methods.

Now that we had an idea of the farmer’s perspective towards fungi, it was time for us to learn the pathologist’s perspective.

Dr. Kelly Turkington: The severity of Sclerotinia sclerotiorum...

Dr. Turkington holds a Masters Degree and Ph.D in Plant Pathology, focusing on the epidemiology of S. sclerotiorum stem rot in canola. For the last 23 years, Kelly has been working as a Research Scientist for Agriculture and Agri-Food Canada at the Lacombe Research Centre in Alberta.

We consulted him to learn more about the progression of S. sclerotiorum fungus, its impact on farmers and preventative and prescriptive measures to combat it.

According to Kelly, the cost to treat fungi like S. sclerotiorum can be around $20-$30 per acre, severely reducing a farmer’s bottom line. To assess risk of fungal growth, farmers employ a checklist by assigning point values to certain factors including the plant, the host and the environment. This is a very broad indication of risk and is not an exact science as there is ambiguity in the checklist. Some companies have started using DNA-based chips to quantitatively determine the percentage infestation of a plant.

In determining if Pheophorbide would be an ideal anti-fungal agent, he directed us to consider the full chemical profile of pheophorbide. Not just it’s effects on fungi but also on non-target organisms. Another consideration is the societal aspect of such a product. Members of the farming community and industry only care if the product is cheap and effective, but society as a whole tends to support products which are “organic” or come from the environment in a responsible manner.

5. Iterate on Design

Further improving our project design

It was important that we continued to revisit our canola contacts and academic advisors to iteratively improve our experimental designs would give the best results to put our solution into industry.

To do this, we hosted a mid-summer faculty talk, inviting all the experts we had consultated with earlier in the project. Additionally, with our tools for the farmers, we met with the Canadian Grain Commission to do testing of our standardized seed grading machine. Our weather prediction model is in peer-review at Alberta Academic Review with goal of being published and made available to the academic community for other researchers to further improve.

Faculty Talk

Once we consolidated and incorporated the advice from our expert consultations with professors, and canola industry members, we were able to design and define the various wet and dry lab projects encompassing yOIL. From here, we decided to host a mid-summer faculty talk to get further feedback on our experimental design and current progress. Supervisors, past iGEM Calgary members, and our business contacts, and the aforementioned professor were also invited to the presentation to give us feedback on the feasibility of our projects.

Overall, we received positive feedback on our dry and wet lab projects. The biochemistry and microbiology professors we invited confirmed that our T7-inducible genetic circuit with BL21 was simple and well characterized for protein production. However, we were informed that our signal peptides may not be as efficient as extracting the protein from the cell lysate, but would be a good future direction to consider. Additionally, we got a few specific comments about the emulsion system on how to improve it where to get more information about how to design efficient emulsions. However, the general use of an emulsion-system was well received and we were commended for considering the steps necessary for its industrial scale-up (we have this right). A few professors also gave suggestions on how to improve the lab experiments we planned for chlorophyll repurposing. We initially thought to add ethanol to the chlorophyll binding proteins to release chlorophyll, but we were given alternative solutions that would instead temporarily weaken the protein’s structure to release chlorophyll. Additionally, the use of a magnesium utilizing protein was an idea given to characterize an enzyme in the chlorophyll degradation pathway.

In conclusion, we were able to take the advice we received from our meetings at the beginning of our project to create clear wet lab and dry lab components each with a defined experimental workflow. The faculty talk was an opportunity for us to concisely present our plans to recieve expert feedback again, and iteratively improve our project design.

Canadian Grain Commission

To verify field implementation of the standardized seed grading project into industry, we went to visit seed grading expertsRomeo Honorio and Scott Kippin from the definitionCanadian Grain Commission (CGC). We met Romeo at where they explained current attempts to standardize seed grading. Our visit allowed us to have large graded seed samples from the experts, that way we can base our grading algorithm on their counts. They also provided significant feedback on standardizing the grading conditions. At the CGC office all the walls were a specific grey, and the grading tables another specific grey, and the light fixtures were at very controlled intensities and highly diffused. These design considerations were embraced by standardized grading machine to allow for a better prototype. Another piece of learned information is that farmers do not care about specific percentages of DGR, only the final grade as that is their paycheck. Grain elevators, oil refineries, and crushers are significantly invested in knowing the exact percentage of the grade as it directly affects the final product canola oil. Even if we started this project by hearing about a farmer's grievance with the grading system, our project would have serious effects throughout the definitioncanola pipeline. So we learned that whilst farmers would benefit from our invention, the other cogs in the canola industry would be more significantly affected by the standardization of DGR grading.

Another piece of information that we learned is that there exists some machines that can grade chlorophyll successfully on the market, but they are usually slow, and cost upwards of 40,000-60,000 dollars which is more than what grain elevators are willing to pay. Romeo liked our machine but wishes it will be taken further both in algorithm quality and user-oriented hardware design, especially in terms of the speed at which it is built. He also emphasized buying the best camera possible as the camera affects the entire project, hopefully an definitioninfrared camera to help us have better readings, they also suggested a future direction of grading wheat as the current methods are even more prone to human error than canola.

Sunny Days Publication

Do we have proof for this somewhere? Is it written somewhere else?

Should it be included here?

Project

Description Wet Lab Overview Chlorophyll Extraction Protein Emulsification Repurposing Chlorophyll Anti-Fungal Demonstrate Results

Human Centered Design

Integrated Human Practices Entrepreneurship Education Collaborations Safety

Modelling

Overview Measurement Software Tools Results

Parts

Parts Overview Collection Improve Characterize Validate

Journal

Entries Protocols Judging Appendix

Team

Members Attributions Sponsors