# Human Centered Design

## How can we solve problems that affect people?

Providing a solution to the green seed problem could be the key to alleviating losses felt by those in the canola industry. Our team’s vision was to make yOIL that solution. In order to do so, we had to ensure that we created something that was effective, usable, and targeted areas which impacted people the most. The only way 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.

yOIL started off small in scope. Nothing more than a process to remove chlorophyll from canola oil. It wasn't until we spoke to everyone involved in the industry that we realized we could do so much more.

This is the story of how yOIL was born from a desire to help oil producers, and grew into an all-encompassing solution through iterative meetings with contacts from all over the industry.

# 1. Understand the Problem

## Key Stakeholder Interviews

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 does it impact? 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 those two, we also identified agronomists and organizational bodies working 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.

## What did we discover?

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. We can think of these stages as part of a pipeline.

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.

Farmers have no tools to plan for unexpected frost or drought, and gamble their harvest every year on unpredictable whether.

Leading to farmers inevitably producing green seeds, which get downgraded and are sold at a lower price.

The process to grade green seeds is not standardized or objective, making the system less fair.

Oil processors are forced to spend more time and money to purify the oil produced from the green seeds they receive, the archaic acid-activated clay purification method is unsustainable.

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.

# 2. Ideate

## Propose solutions and verify their need

Having identified and understood the multiple adverse aspects of the green seed problem, our team began brainstorming solutions.

After discussing and analyzing our stakeholders' needs, we decided on two synthetic biology solutions and one engineering based solution. These are all aimed at the oil processing stage where current methods are expensive, result in oil loss, and are not environmentally friendly.

To discover more about these solutions, click on the buttons below.

The above solutions focused on the oil processing stage. However, after hearing stories from farmers like Craig about their losses due to green seed, we were determined to help them as well.

Preventing green seed entirely would be the most beneficial solution for farmers, but we knew that was infeasible in our time frame. We determined that we could help them by creating software and hardware solutions that help with crop management and seed grading.

To learn about these software and hardware solutions, click on the buttons below.

## Verifying need - Canolapalooza

Midway through the summer, we were lucky enough to be able to attend canolaPALOOZA. An annual event held in Lacombe, Alberta, canolaPALOOZA is the “agronomy event of the summer” where canola experts from all over the prairies come together to discuss all things canola. Everyone from farmers, agronomists, mycologists and even an undergraduate iGEM team from the University of Calgary descended on a lone canola field in Leduc to talk shop.

Many of our initial contacts, including Ward Toma and Dr. Barthet, were in attendance, in addition to many others. It was an excellent opportunity for us to propose our ideas to a wide cross-section of the canola industry, and get feedback on the commercial viability of our solutions. The event was extremely valuable because this is where we learned about the fungal infestations currently affecting canola crops in Alberta. A fortuitous meeting which would give us the inspiration to repurpose chlorophyll into an anti-fungal agent.

This event lead to a connection with the Canadian Grain Commission, who expressed their interest in the standard seed grading system. They informed us that there had been a similar project in the past that was successful, but got scrapped due to its cost. This inspired us to make our standardized grading machine as inexpensive as possible.

# 3. Design Solutions

## Expert consultations

With the need for our solutions verified, it was time for us to put shovels in the ground ( or bacteria on the agar) and begin designing yOIL. Our development would be agile and adaptable. Continually meeting with stakeholders and integrating feedback throughout the design process to ensure yOIL holistically embodies the needs of everyone involved in the canola oil pipeline.

## 1) Chlorophyll Extraction System

The clay method is not reusable, environmentally friendly, or selective to chlorophyll, thus, we needed to come up with an improved solution. 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 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.

## 2) Chlorophyll Repurposing

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 (Xodo et al., 2012) and anti-fungal treatments (Liang et al.,2016). Seeing how valuable the product could be, 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.

## 3) Tools for the farmers

Craig, Angela, and Ward, stressed two major hurdles farmers face when they produce and sell their seeds to oil processors: unpredictable weather and unstandardized seed grading practices. We turned to computation as a solution to address both problems. Current weather forecasts cannot predict weather 3-4 months into the future, preventing farmers from employing agronomic techniques that could save their crops. This drove us to design a long-term weather prediction system to give farmers the tools they need to make the right choices.

The current seed 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. Higher green seed percentage means that a farmer's harvest is sold at a discount to grain elevators, effectively taking money out of their pockets. 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. Not just any machine. A mean green machine .

## 4) Pheophorbide

Attending CanolaPALOOZA allowed us to evaluate the need for 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. Fungal species such as Sclerotinia sclerotiorum is "one of the most serious and damaging diseases of [canola seed]" (Garg et al., 2010). We saw an opportunity here. An opportunity to repurpose the chlorophyll captured from canola oil and convert it into pheophorbide for use as an anti-fungal agent. But first, we needed to investigate how pheophorbide can work as an anti-fungal.

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.

# 4. Evaluate and Iterate on Design

## Further improving our project design

We are dedicated to improving our solutions to serve the people in our community. We involved our stakeholders and academic advisors in every stage of our solutions. From investigation and ideation, to prototyping and testing, yOIL's design process was purely human-centered.

We hosted a mid-summer faculty talk, inviting experts we consultated with earlier in the project, to close the loop with them. Additionally, with our tools, we met with the Canadian Grain Commission to perform testing of our standardized seed grading machine. Our weather prediction model, Sunny Days is undergoing peer-review at Alberta Academic Review with the goal of being published and made available to the academic community for improvement.

## 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 iterative feedback on our experimental design and current progress. Supervisors, past iGEM Calgary members, and business contacts, were also invited to the presentation to provide their unique perspectives.

Overall, we recieved positive feedback and constructive criticism on all aspects of yOIL. Biochemists and microbiologists in attendance confirmed that our T7-inducible circuits in BL21(DE3) E.coli was the way to go. However, we were informed that using signal peptides won't be as efficient as extracting proteins from cell lysate, but is a good consideration for future scale-up. The idea to use an emulsion system was well recieved as a workaround for preventing our protein from denaturing in oil, and we were commended for considering future industrial scale-up in that regard. Initially, we thought to add ethanol to the chlorophyll binding proteins to release chlorophyll, but we were given an alternative which would instead temporarily weaken the protein's structure, thus releasing chlorophyll.

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.

To verify field implementation of the standardized seed grading project into industry, we went to visit seed grading experts Romeo Honorio and Scott Kippin from the Canadian Grain Commission (CGC). We brought along our first prototype for Mean Green Machine (MGM) to get feedback on our hardward implementation.

We got large graded seed samples from the experts themselves to see how our grading algorithm stacks up. Seeing the highly standardized environment where seeds are graded, muted grey walls and tables to control light diffusion, gave us more design considerations regarding Mean Green Machine's first prototype. More importantly we learned that farmers do not care about specific percentages of distinctly green seed (DGR), only the final grading which also indicates how green their paycheque will be.

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 canola pipeline. 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 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 infrared camera to give better readings. The also gave us the idea to apply MGM to grading wheat, an excellent future direction to reduce the human error present in that sector.

# References

1. E Xodo, L., Rapozzi, V., Zacchigna, M., Drioli, S., & Zorzet, S. (2012). The chlorophyll catabolite pheophorbide a as a photosensitizer for the photodynamic therapy. Current medicinal chemistry, 19(6), 799-807.

2. Liang, Y. I., Lu, L. M., Chen, Y., & Lin, Y. K. (2016). Photodynamic therapy as an antifungal treatment. Experimental and therapeutic medicine, 12(1), 23-27.

3. Garg, H., Atri, C., Sandhu, P. S., Kaur, B., Renton, M., Banga, S. K., ... & Banga, S. S. (2010). High level of resistance to Sclerotinia sclerotiorum in introgression lines derived from hybridization between wild crucifers and the crop Brassica species B. napus and B. juncea. Field Crops Research, 117(1), 51-58.