Team:Calgary/Human Practices
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 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.
Design Process:
- 1. Investigate to Understand the Problem
- Design Solutions
- Propose Solutions
- Evaluate
- Iterate on Design
1. Investigate to 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 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.
2. Design Solutions
Expert Consultations
What did we make
What did the people think?
Chlorophyll Extraction System
We knew that we needed to design a system that utilized a protein to capture chlorophyll, but we weren’t
confident in our initial design. We were stumped as to which chlorophyll-binding
protein we should 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 five protein bio-chemists and a micro-biologist who gave us insight
into how to design our system.
Dr. Gordon Chua
Choosing the right chassis...
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. 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. 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. 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.
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.
Discoveries:
What is it?
Who's impacted?
How bad is it?
How this impacts design:
3. Proposing Solutions
Revisiting our stakeholders
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Pheophorbide
pheophorbie hp is important because... our reasoning.
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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 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 Sclerotinia sclerotinium, 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 Sclerotinia.
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.
We consulted him to learn more about the progression of Sclerotinia fungus, its impact on farmers and preventative and prescriptive measures to combat it.
According to Kelly, the cost to treat fungi like Sclerotinia 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
To assist in the dynamic characterization by other teams we looked to develop a methodology that allows for the calculation and aggregation of Brownian motion measurements for each amino acid in a sequence. The Brownian motion measurement chosen was the Root Mean Square Fluctuation(RMSF) calculated for every atom of a protein in ten picosecond intervals.
The RMSF data was calculated from a nanosecond Molecular Dynamic Simulation(MDS) completed within GROMACS, an industrial MDS software. These values were then averaged over each amino acid, this ensured that the unit of measurement was observed on a scale that was modifiable by teams. This resulted in a series of curves that quantitatively expressed the dynamics for each amino acid.