<p>Throughout our project there were several instances where we underwent iterations of the <b>engineering design process</b>. The engineering design process includes designing, building, testing and then improving a design. This process is repeated until the goal of the project has been reached or the most ideal version of the design has been achieved.</p>
<br><br>
<figure>
<imgsrc="https://static.igem.wiki/teams/4296/wiki/images/eng-success.png"alt="Four Stages of Engineering Success">
<p>To test the expression of our genetic constructs in wheat our team decided to use wheat protoplasts. Protoplasts are plant cells where the cell walls have been enzymatically removed, making them easier to transform with foreign DNA. One challenge our team faced was optimizing our protoplast isolation procedure. This is one example of an instance where our team utilized the engineering design process.</p>
<p>The goal of the protoplast isolation procedure was to be able to successfully grow wheat plants and isolate enough protoplasts from clippings of their leaves to be able to perform proof of concept transformation experiments with our genetic constructs. Early on our team realized that this would be a challenging process. We knew that it could take a long time to optimize a new protocol, and there was limited time in the competition. </p>
<p>After going through five rounds of improvement of our procedure, we were successful in generating viable and sufficient amounts of protoplasts.</p>
<p>We started by looking through the literature to find protoplast isolation protocols. We compared different protocols to find a consensus on reagents and steps. We also asked our advisors who had experience isolating protoplasts for their feedback on the protocols we had found. Eventually the protocol we decided on was one used to make wheat and barley protoplasts successfully in a lab at UBC. </p>
<br><br>
<h3id="IterationOne">Protocol Iteration #1</h3>
<hr>
<p>This is the protoplast isolation procedure we used for our first trial run of making protoplasts</p>
<ol>
<li>Grow about 50 wheat seedlings for 7 to 10 days.</li>
<li>Collect all leaves from the seedlings. Wash them and store them in 2x sterile dH2O in 50ml Falcon tubes.</li>
<li>Cut slits in leaves using a sterile scalpel (longitudinally, 0.5mm strips).</li>
<li>Place cut leaves in 13ml culture tubes and fill it with cell wall enzyme solution (~ 8-10 ml per tube) and leave them in the dark at room temperature for 4-5 hours with gentle shaking (50 rpm). Make sure temp is not > 25C.</li>
<li>Very <i>gently</i> swirl the tubes during the incubation time in order to gently move the leaf tissue around in the enzyme solution to release the protoplasts. </li>
<li>Always use wide bore tips or 5ml pipette (has wide bore) to pipette protoplasts.</li>
<li>After 4 hours, gently pipette the protoplasts into a 13 ml tube (culture tube) to a total vol of 10 ml (or fill to 8 ml and complete to 10 ml with washed leaf sucrose).</li>
<li>Overlay the solution with 1 ml of W5 media by adding gently to the top of the solution (Do not mix). Spin the tubes at 250 g (~ 930 rpm), 10 min, no brake.</li>
<li>Remove the protoplasts from the boundary (middle layer) using a pipette and place them into a new 13 ml tube (it is ok if I take a bit of other layers) (In this step 3 layers are formed, on top is W5, green protoplast in middle and enzyme solution along with debris and dead cells on bottom).</li>
<li>Add gently W5 to a total volume of 5 ml (volume of protoplasts is >2-3 ml/tube). [Want to remove cellulase solution]. Invert the tube gently a couple of times and spin at 250 g, 5 min, no brake.</li>
<li>Remove the liquid on the top (top is W5 and protoplast will be in bottom) and gently suspend protoplasts in ~ 5 ml W5 by inverting tube gently (At this stage several tubes can be pooled).</li>
<li>Incubate them at 4C (fridge) for at least 30 min.</li>
<li>Count the protoplasts. Want 4-5 x 106 per ml for transfections.</li>
</ol>
<p>The first time we tried to make protoplasts we used the protocol given to us by a lab at UBC without any modifications or changes. The wheat plants used for the protocol grew successfully. 15 plant leaves were used for the protoplast isolation.</p>
<figure>
<imgsrc="https://static.igem.wiki/teams/4296/wiki/images/first-protoplast-day-6.jpg"alt="6-day old wheat seedlings">
<imgsrc="https://static.igem.wiki/teams/4296/wiki/images/first-protoplast-day-7.jpg"alt="7-day old wheat seedlings">
<figcaption>(Left) Wheat seedlings used for first protoplast isolation trial, photo of 6-day old wheat plants, (Right) 7-day old wheat plants from same trial.</figcaption>
</figure>
<p>However, no protoplasts were successfully isolated from the clippings of the wheat leaves.</p>
<br><br>
<h3id="IterationTwo">Protocol Iteration #2</h3>
<hr>
<p>Considering the results from the first trial of protoplast making, improvements were planned to the protocol:</p>
<ol>
<li>We found that the wheat seeds planted at 4.5cm depth sprouted more than those planted at 2cm depth, so for future trials all seeds will be planted at 4.5 cm depth.</li>
<li>Only 15 leaves were used for this protoplast isolation. For future protoplast isolation trials more will be used. Plan to start the next trial with 30-40 leaves.</li>
<li>During cutting of the wheat leaves, try as hard not to cut through the entire leaf and just make very small slits, this may reduce debris and dead protoplasts.</li>
<li>When adding W5 for the first time, add directly to a falcon tube instead of a culture tube so that they don’t mix.</li>
<li>Will only use p1000 pipette/serological pipettes moving forward since protoplasts are so delicate and tear easily.</li>
<li>Will wash the leaf debris post incubation with 0.5M sucrose. After each wash the 10 ml aliquots of the solution will be taken and added to culture tubes.</li>
</ol>
<p>Using the modified protoplast isolation procedure for the second trial run of making protoplasts, again the wheat plants used for the protocol grew successfully. We had a higher percentage of seeds sprout when they were all planted at the 4.5cm depth. 33 wheat leaves were used when making protoplasts.</p>
<figure>
<imgsrc="https://static.igem.wiki/teams/4296/wiki/images/second-protoplast-day-6.jpg"alt="6-day old wheat seedlings from second trial of protoplast isolation">
<figcaption>Photo of 6-day old wheat seedlings from the second trial of protoplast isolation.</figcaption>
</figure>
<p>Very few protoplasts were made from the clippings of the leaves of the plants, and those made were fractured and torn. The photo of the protoplasts can be seen below:</p>
<figure>
<imgsrc="https://static.igem.wiki/teams/4296/wiki/images/second-protoplast.jpg"alt="Second round of protoplast isolation">
<figcaption>Photo of protoplasts isolated from the second round of trying to isolate protoplasts. Only 4 protoplasts (green circles) are seen under the hemocytometer.</figcaption>
</figure>
<br><br>
<h3id="IterationThree">Protocol Iteration #3</h3>
<hr>
<p>Considering the results from the second trial of protoplast making improvements were planned to the protocol:</p>
<ol>
<li>Start the protoplast isolation immediately after harvesting the leaves. Waiting overnight could dehydrate the leaves and cells could die, reducing protoplast yields. Keep the cut leaves in deionized water after harvesting if there is a delay before the next step of the protocol.</li>
<li>Resuspend the protoplast solution in only 1ml of W5 solution instead of 5ml to concentrate them more.</li>
<li>Be gentler with mixing and centrifuging since the protoplasts are very delicate and easily burst. </li>
<li>Do not store cut leaves in cell wall enzyme solution overnight at a temperature greater than 25 degrees celsius. Higher temperatures can cause the cells to die.</li>
</ol>
<p>In the third trial run of making protoplasts, no protoplasts were obtained using the modified protoplast isolation procedure. What can be seen is torn apart plant cells.</p>
<figure>
<imgsrc="https://static.igem.wiki/teams/4296/wiki/images/third-protoplast.jpg"alt="Third round of protoplast isolation">
<figcaption>Photo of protoplasts isolated from the third trial of trying to isolate protoplasts. No intact protoplasts visible, many of the plant cells are sheared and their organelles and chloroplasts are visible.</figcaption>
</figure>
<br><br>
<h3id="IterationFour">Protocol Iteration #4</h3>
<hr>
<p>Considering the results from the third trial of protoplast making improvements were planned to the protocol:</p>
<ol>
<li>Plant about 60 seedlings per protoplast trial as only about 50 will germinate and sprout. 50 seedling leaves are ideal for the protoplast isolation</li>
<li>Cutting the wheat leaves latitudinally in 0.5mm strips rather than longitudinally. The slits are made perpendicular to the midrib of the leaf. Dicing the leaves as much as possible increases surface area to allow thee enzyme solution to work better.</li>
<li>Let leaves soak in mannitol for 5-10 minutes after cutting. Mannitol helps plant cells to tolerate salt stress, also can be used as food so protoplasts can metabolize mannitol during the digestion phase</li>
<li>For step 11 do 1 ml of W5 instead since it is better to have a more concentrated solution than a less concentrated solution of protoplasts). Place on ice to cool (this allows faster cooling to 4C).</li>
</ol>
<p>In the fourth run of making protoplasts very few protoplasts were obtained, however these protoplasts were not burst or damaged. This improvement indicated that the changes made to the protocol were beneficial in bringing us closer to our end goal.</p>
<figure>
<imgsrc="https://static.igem.wiki/teams/4296/wiki/images/fourth-protoplast.jpg"alt="Fourth round of protoplast isolation">
<figcaption>Photo of protoplasts isolated from the fourth trial of trying to isolate protoplasts. A single intact protoplast can be viewed with a few sheared cells.</figcaption>
<p>After the fourth trial of protoplast making with little success previously. We went back to the literature and incorporated changes we found in other protoplast procedures. The following improvements were planned to the protocol:</p>
<ol>
<li>Make the reagents on the same day as making protoplasts do avoid degradation of enzymes in the reagents such as the cell wall enzyme solution</li>
<li>Reduce enzyme content in cell wall enzyme solution by 50% as many protoplasts were degraded in previous trials</li>
<li>Remove the sucrose wash step</li>
<li>Perform 3 W5 wash and concentrate steps with a 30 minute incubation on ice after each resuspension of the protoplast pellet. </li>
</ol>
<p>In the fifth run of making protoplasts we successfully made many protoplasts! We made protoplasts of good quality and in sufficient amounts that we could proceed to use them in transfection experiments.</p>
<figure>
<imgsrc="https://static.igem.wiki/teams/4296/wiki/images/fifth-protoplast.png"alt="Fifth round of protoplast isolation">
<figcaption>Photo of protoplasts isolated from the fifth trial of trying to isolate protoplasts. Many intact protoplasts are visible and some cells have been sheared.</figcaption>
<p>Synaestivum, if developed into a product, would be sold as transgenic seeds that grow into wheat expressing enzymes to battle heat stress when experiencing high temperatures. In order to evaluate the feasibility of our project’s implementation, we carried out market research and developed implementation plans described in the sections below, as well as increased the entrepreneurial capabilities of our team through consultations, meetings, and learning.</p>
<br><br>
<h2id="MarketResearch">Market Research</h2>
<hr>
<h3>Identifying Unmet Needs and Potential Customers</h3>
<hr>
<p>Since Synaestivum’s inception , we have aimed to solve farmers’ most pressing climate-based issues. Our evidence-based perspective was that heat was the most pressing climate-based factor affecting yields. In order to investigate potential niches for our product, we conducted market research on the needs of potential customers to confirm this stance. In our vision, <b>customers would benefit from increased productivity, resulting in higher financial returns for the same amount of effort</b>. This would be possible due to increased yields resulting from lower heat stress crop loss. <b>Decreased biomass loss would increase agricultural efficiency</b>, thereby <b>lowering the carbon footprint</b> of modified crops relative to unmodified crops.</p>
<p>In order to identify unmet needs, specifically climate-based needs, we were able to liaise with farmers that could potentially resonate with the issue we are aiming to address. In our customer discovery, we were aiming to obtain opinions from varying perspectives; therefore, we conducted consultations with representatives from both Canadian and Indian farmers.</p>
<p>We carried out three separate visits to two of ten wheat farms in British Columbia, whose crops were presumably negatively impacted by the heat waves of 2021, as well as a produce farm whose produce was reportedly positively impacted by the heat. As discussed in our Integrated Human Practices, two of the three farmer representatives (66%) expressed that <b>increased temperatures were the climate-based factor that most severely impacted their yield over the years</b>, while one mentioned that drought was instead, due to him being a small-scale farmer that did not have access to irrigation. All three farms were led by individuals who were born into farming families and therefore had decades of experience, therefore their perspectives were weighing in on decades of observing climate-based factors affect crop yield. All three farmers expressed that <b>they would purchase our seed product if it became available due to the trends they have observed over time, demonstrating support from our first potential customers</b>. This was backed by Kelly Boles, crop consultant that has provided advice on variety selection and growing parameters to Canadian farmers for multiple decades. He mentioned that the global need will be for heat-resistant grains, while locally in Western Canada he observes more of a need for drought-resistant grains at the moment. He expressed support of our initiative, especially due to its consideration of increased yield in addition to heat resistance, and is therefore a contact through which we could obtain a customer stream.</p>
<p>While this customer-based insight was useful for assessing unmet individual needs, we were able to assess market gaps at the nationwide level. In speaking to Bioceres, the first company to have FDA-approved genetically modified (GM) wheat, they mentioned certain markets that their current solutions cannot reach. <b>They developed drought-resistant wheat, which works effectively in environments of increased temperatures hence leading to their current approvals, but does not reach countries that have high precipitation and high temperatures</b>. They gave the example of Brazil, where high precipitation removes the need for drought-resistant crops, but they do have a need for crops that withstand high temperatures. Since there are only drought-resistant wheat crops in the process of approval for sale in the public market at the moment, this identifies nationwide needs that are currently not addressed by technologies in development, posing a good opportunity for Synaestivum to benefit these communities.</p>
<p>These identified needs are reflected in broader global trends: as both world population and scarcity increase, there is a universal pressure on companies to become greener. Our participation in SynBio Canada 2022, where startups and investors shared insights on trends in the synthetic biology industry, reinforced the utility of our project. Caitlyn Walsh, Managing Director of Thematic Investment at CPP investments, argued that <b>despite the pressure on companies to integrate sustainability into their activities, there is a lack of greener products that exist for companies to use towards their sustainable initiatives. Our product is considered more environmentally sustainable since it results in less yield loss due to heat, therefore utilizing the same resources more efficiently</b>. It is hence a greener product for organizations such as seed banks, flour milling companies, and bread production companies to utilize to reach their sustainability goals.</p>
<br><br>
<h3>Identifying Stakeholders</h3>
<hr>
<p>Apart from liaising with potential customers to discover and understand the issues they face, meetings like the one with Kelly Boles were able to provide insight into the stakeholders that could affect or be affected by our venture. In order to accurately depict in what areas of our commercialization journey stakeholders would come in, we developed the following production to customer map that identifies where stakeholders have the opportunity to intervene. </p>
<p>Given that the success of our commercialization would largely depend on pre-production requirements such as funding opportunities and the trends observed in agricultural technologies, we spoke with Carla Berquo, Director of the Ag-Tech engine in Bioenterprise, a Canada-based AgriTech accelerator. While she mentioned that the fastest growing technologies in Canadian agriculture were based around automation and robotics, she highlighted that <b>a product solving an important problem often receives emphasis in evaluation by accelerators and other funding entities upon evaluation. According to Carla, genetically modified organism (GMO) technologies are not at a disadvantage when applying for funding, since many funding entities (including Bioenterprise) work with the customer to advocate for the technology to the government to be able to decrease policy-based barriers</b>. She expressed support and enthusiasm for the project, and put us in contact with a BC-based accelerator and an advisor for Bioenterprise for future help with funding and GMO approvals. This support from an investment entity increases the credibility of the funding aspects of our project, outlined in more detail in section 3.2 of this page, while also reassuring the low barrier that our product being GMO-based has in terms of putting this into market.</p>
<p>In order to measure the influence and interest our stakeholders have in our venture, we <b>performed a stakeholder analysis to locate all of the abovementioned stakeholders within these parameters</b>. Those that have high influence and interest on the success of our venture, such as legal policy individuals and investors, require the highest level of maintenance and monitoring, and identifying allows us to focus our efforts on keeping them satisfied. Groups that have low influence, but high interest, such as farmers, we should keep informed and liaise with to not undermine their needs. On the other hand, groups that have high influence and low interest should be monitored and kept with the level of information they are comfortable with. Being able to identify these stakeholder categories paves the way for different modes and frequencies of communication that should occur with each one, as well as focuses the team’s energies in anticipating stakeholder reactions to our decisions and development. <b>We began exercising the results of our stakeholder analysis through prioritizing communicating with and informing our stakeholders of highest influence and interest, resulting in the conversations outlined in the previous section with farmers, the accelerator Bioenterprise, and a main competitor</b>. The support these stakeholders have expressed, and moreover the support in different quadrants of interest and influence, speaks to the credibility of developing our solution further with the strength of our supporting stakeholders.</p>
<p>Synaestivum, has three facets of value that have not been previously combined by companies reaching similar efforts. The heat-inducible expression of SBPase is proposed to increase overall crop yield, therefore competing with producers of value-added wheat that similarly increase yield through non-GMO methods. Synaestivum also competes in the space of producing genetically modified wheat for the purpose of heat resilience, and thirdly in the broader space of modifying globally relevant crops used for human feed for increased heat tolerance. We see it as a stronger competitor due to it working with common wheat due to how engrained this food product is in our diets, making it difficult to replace with for example heat-resilient rice if wheat became too sensitive to differing temperatures. Due to the multifaceted approach of the project, we decided to conduct a petal competitive analysis to pinpoint how and why we are able to offer a solution of increased value than other options. This involves creating one ‘petal’ for every distinct competitive space that the product is in, placing it in the middle of these differing markets.</p>
<p>This petal analysis allowed us to separate our competitors by category, therefore <p>identifying the quality required of each of our product’s features to surpass our competitors</p>. For example, if we observed that the companies developing wheat with increased yield had products with higher quality than those creating wheat with increased heat tolerance, this <b>sets a higher threshold to strive to reach with our results</b>. An interesting outcome is that many of the entities developing heat-resilient global crops for human feed - for example, Chinese Academy of Sciences developing heat-resilient rice - are being developed in universities, which might have more stable sources of funding than the startup company our product would be developed by. However, this also means that they would have reduced capabilities for scaling up the processes due to the scale of university work, putting us at an advantage during the scale-up stages of our product development. The second outcome is the realization that our product is the only one that integrates all three competitive spaces to offer an added value than other companies. Our ability to work in the increased yield space has notable value due to this being the main parameter farmers select for, according to the crop consultant Kelly Boles. He says that choosing grain varieties to plant based on climate-based parameters comes secondary to yield. Our double strategy and design in both climate-based and yield-based value puts us in a good space in the market as emphasized by this competitive analysis.</p>
<br><br>
<h3>Market Sizing</h3>
<hr>
<p>To explore the monetary value of our project, the size of our market and its estimated revenue was established. Defining our market size will allow for potential stakeholders and organizations to estimate the profit from our product, as no predefined quantitative value exists due to its novelty. Identifying our core market will also aid in the development of an effective marketing strategy that is specifically targeted for our customer base. The size of the total addressable market (TAM), service addressable market (SAM), and the service obtainable market (SOM) were defined, and the estimated revenue for the Synaestivum was generated using the SOM assumptions.</p>
<p>The TAM is obtained from the 135 million farms globally, which is valued at $731 million USD, generating $155 billion USD annually. (Vantage Market Research, 2022) The SAM is a subset of this TAM, which includes the farms that are currently experiencing crop yield loss due to extreme heat, and would be able to accept a transgenic variety as a solution. As the northern hemisphere is currently experiencing extreme heat waves due to climate change, wheat producing farms in China, India, Russia, the United States, Canada, France, Pakistan, Ukraine, Germany, and Turkey would benefit from our heat tolerant variety. (cite) Of these countries, only India, the United States, and Canada are able to accept our transgenic strain as a solution as genetically modified crops are regulated in the remaining nations and laws prohibit farmers from growing our product. (cite) Our SAM includes 200,000 wheat farms in the United States and Canada, and 53.1 million in India. (cite) The SOM for our product represents the portion of our SAM that is likely obtainable given our project’s scope and current resources. The initial reachable customer base is expected to be Canadian farms, where farmers have been growing other types of genetically modified crops such as corn, canola, and soybeans for over two decades. (cite) Canadian wheat farms produced 35 million tonnes of wheat in 2022 across 24 million acres, which defines our SOM by area. (cite,cite) </p>
<p>With insight from farmers and crop consultations in British Columbia, Alberta, and Ontario, we obtained the average market price for wheat seeds to be $23 USD per acre, resulting in a market size by gross revenue of $552 million USD for our SOM. The initial market for our product is predicted to be farms in British Columbia and Alberta given our location, which makes up 39% of wheat farms in Canada. (cite) Within this percentage, an initial penetration rate of 10% was estimated, resulting in a total rate of 3.9% for the SOM. By multiplying the market size by revenue of $552 million USD with the penetration rate, we obtained the market size by gross revenue of Synaestivum to be $21.5 million USD. This market size allows for our team to couple a monetary value with our product for stakeholders, and has the potential to grow as the demand for our solution increases with the rising climate continuing to affect crop yields globally.</p>
<p>We conducted a SWOT analysis to determine if our current design and planned activities are a feasible way of moving forwards.</p>
<table>
<tr>
<td></td>
<td><b>Positives to be exploited</b></td>
<td><b>Negatives to be minimized</b></td>
</tr>
<tr>
<td>Internal factors</td>
<td>
<b><u>Strengths</u></b>
<ul>
<li>Our team members have high industry expertise and motivation, as well as various contacts in the industry that could serve useful</li>
<li>Scale-up procedures for genetically modified seeds has been well validated, as opposed to scale-up processes for synthetic biology innovations that use less characterized host organisms and final products.</li>
</ul>
</td>
<td>
<b><u>Risks</u></b>
<ul>
<li>Lack of business and legal expertise; we would hire a business and legal team member</li>
<li>Distance to market; our timeline is a long one before this would become a profitable business.</li>
</ul>
</td>
</tr>
<tr>
<td>External factors</td>
<td>
<b><u>Opportunities</u></b>
<ul>
<li>Partnerships with more experienced agricultural biotech companies, such as Bioceres</li>
<li>Increasing global market for genetically modified foods, and high approvals in Canada, increase</li>
<li>Developing parallel products for other crops of relevance; this could become a threat if developed in a market where there are more competitors</li>
</ul>
</td>
<td>
<b><u>Threats</u></b>
<ul>
<li>Increasing competition due to increased acceptance and market size for genetically modified seeds</li>
</ul>
</td>
</tr>
</table>
<br><br>
<p>The evaluation obtained from our SWOT suggests moving into new markets is feasible. However, the increased competition in the genetically modified seeds market is risky if we become a company with a single technology pending approval due to high chances of others developing similar products. Expanding into heat resistance for other crops would be feasible due to high GMO approvals in Canada and an increasing market for genetically modified seeds, and would de-risk the commercialization of the technology. This has been integrated into our timeline (see below).</p>
<br><br>
<h3>Scaling our Innovation</h3>
<hr>
<p>The process of developing Synaestivum to be a commercially available product can be separated into two components: a research and development stage to produce the initial batch of transgenic seeds, then a breeding and harvesting stage where our product will be mass produced to match the demand from our previously determined market size. The initial stage would take place in a wet-lab environment, and involves the genetic transformation of wheat by the agrobacterium method or particle bombardment. These two options are accessible, cost effective, and have an established methodology for our team to follow. (cite, cite) Upon successful transformation, the initial batch of transgenic seeds harvested from the lab would be carried over to the breeding and harvesting stage to take place at a test field. The major costs of production arise from obtaining a laboratory space, greenhouse, and test field. The average commercial rent in Vancouver, where our team is based, is $50 CAD per square foot per year. (cite) For a 1000 square feet lab space, the estimated cost of leasing a lab space is $50,000 CAD. Taking into account the cost of renting specialized laboratory equipment such as lab benches, freezers, and temperature controlled rooms, this cost will be increased by 40%, resulting in the cost of $70,000 CAD per year to obtain a space for research and development. To estimate the cost of obtaining a test field, the average cost of leasing farm land was calculated by multiplying the rent to price ratio from the ‘FCC Farmland Values Report’ by the average value per acre.(cite, cite) In Greater Vancouver, the cost to lease two acres of farmland is $58,000 CAD annually. In addition, the labor costs of a team consisting of two research scientists, three research assistants, and one lab technician is around $310,000 CAD annually, based on average Vancouver salaries for each position. (cite, cite, cite) The yearly cost of our product development is estimated to be around $380,000 CAD for the research and development stage, and $368,000 CAD for the breeding and harvesting stage, based on land and labor costs alone. This can be compared with our market value of $21.5 million USD which is equivalent to $29 million CAD.</p>
<br><br>
<h3>Credibility & Capabilities</h3>
<hr>
<p>Within countries that could develop such agricultural innovation, Canada is especially poised to succeed in these kinds of innovations. Our country has the <b>fourth largest land area dedicated to growing genetically modified crops in the world</b>, and has the <b>third highest number of genetically modified organism approvals</b> (ISAAA Briefs). Canada grows six genetically modified (GM) crops (canola, corn, soy, sugar beet, alfalfa, and atlantic salmon) already, with multiple other GM crops imported into the country. Especially given that some of these crops are grown for direct human consumption, telling of the level of comfort with inserting GM crops into the human food industry, we believe that our project would be easily insertable into the Canadian bioeconomy. Due to the general acceptability of Canada towards genetically modified crops, the development and implementation of our project therefore has support for both production scale-up and policy. As such, our Canada-based team will have a distinct advantage in developing this technology. </p>
<p>In parallel to working on our project, our team members have been working to develop the capabilities to begin this entrepreneurial journey through various workshops and skill-building events. We attended SynBio Canada 2022, in which <b>our team members gained insight on industry trends around synthetic biology</b>. Given the high emphasis on climate solutions being on the rise in biotechnological innovations, this <b>strengthened our belief in branding our technology as an aid in the current climate crisis to ensure food security, which we have implemented throughout our project presentation and value proposition</b>. Connections from VC investors at the conference led us to the list of investors that fit our product in section 3.2 of Funding Plan below, while insights from speakers have contributed to the development of various sections of this page.</p>
<br><br>
<figure>
<imgsrc="https://static.igem.wiki/teams/4296/wiki/images/synbio-canada.jpg"alt="iGEM Team presenting at SynBio-Canada Conference">
<figcaption>iGEM Team presenting at SynBio-Canada Conference</figcaption>
</figure>
<br><br>
<p>Our <b>conversations with founders of successful synthetic biology-based startups, such as Liven Proteins, Future Fields, and Forest City SynBio, have emphasized skills and focuses required to launch our own ventures</b>. Fei Luo, founder of Liven Proteins, emphasized the utility of developing the business-based pitches before expending costs and energy into building the scientific proof of concept; this was especially valuable since the scientific work our team is used to is usually based on laboratory-based proof of concept first. It propelled the thorough development of our business plans through this page. She also challenged our value proposition, pointing out that individuals could switch to other carbohydrates like rice if wheat yields would get too low, leading to us include this in our value proposition plans in section 1.3.</p>
<p>Ela, VP of Product at Future Fields, mentioned to us to not worry about public perceptions of GM foods, which we initially perceived as one of our main barriers in developing this technology to commercialization. Ela believes that with big enough issues at hand, such as climate change, people will see themselves forced to see change as solutions. This perspective about GMO perceptions was strengthened by our talk with Bioenterprise mentioned earlier, where incubators and accelerators offer services to facilitate GMO policy barriers through advocating for the company to the government.</p>
<p>Co-founder of Forest City Synbio, Samir Hadamache, offered insight that highly influential stakeholders like investors initially look at the team’s capabilities, prompting us to outline our teams’ skills and capabilities in the present section. <b>He recommended developing timeline, market sizing revenue numbers, and cost of production, inspiring our development of these items on this page</b>. Furthermore, he suggested monitoring potential customers closely and emphasizing the ability to ensure a customer as much as possible. This led to conducting our stakeholder analysis, where customers and end-users are tabulated in the section to show thorough consideration of their needs.</p>
<p>To not only receive advice on our product development in itself but also leadership capabilities, we attended an event by iGEM’s Women in STEM Initiative. We believe this was instrumental to building our capabilities since 5/7 of our current team’s leadership positions are taken by women. We also consulted with iGEM EPIC Co-Chair Vasiliki Kavvatha on skills to improve as a leader and entrepreneurial exercises to carry out to reach this goal. Her insights resulted in the development of multiple sections of this page such as timeline and market sizing.</p>
<p>Since there are currently regulatory approvals in place that delay our commercialization, a significant portion of our project timeline integrates legal timeline in parallel to proof of concept and product development timeline. Genetically modified organisms need to be approved by Health Canada, and additionally by the Canadian Food Inspection Agency since this is a Plant with a Novel Trait (PNT) being used for human feed. Both regulatory processes require pre-application experiments and conduct trials to confirm function and prevent contamination in the field. Pre-application experiments must demonstrate the consequences of gene flow in the system, altered plant pest potential, and impact on non-target organisms. After government evaluation based on the molecular, genetic and field testing, the PNT is approved for use and commercialization plans can ensue.</p>
<figure>
<imgsrc="https://static.igem.wiki/teams/4296/wiki/images/wistem.png"alt="iGEM Team attending WiSTEM conference">
<figcaption>iGEM Team attending WiSTEM conference</figcaption>
</figure>
<br><br>
<h3>Funding Plan</h3>
<hr>
<p>As Fei Luo emphasized to us, the business-based feasibility and ensuring financial stability is the priority in commercializing a scientific technology, more than the technicalities of how to fulfill the value proposition. Consequently, <b>we have identified sources of potential funding to know where and how to focus the pitching efforts of our value proposition and make this the focus of our early entrepreneurial efforts</b>. The emphasis on finding funding opportunities that would be used throughout the whole life cycle of our company comes from insight from Alison Sunstrum, venture partner at Builders VC. At SynBio Canada, she emphasized the need for solid funding infrastructure past the early stage in Canada, specifically given that more funding is usually given at the academic level. Our <b>search and identification has removed funding opportunities that cover aspects of agriculture we are not addressing</b> - for example, Cultivator’s AgTech Accelerator - <b>while keeping opportunities that would fit our product at different stages of development and Technology Readiness</b> (Cultivator).</p>
<p>At our initial stages, we would both ensure funding and increase our skills through enrolling in pitch competitions and incubators like Falling Walls (application for a 3-minute pitch competition has been already sent) and Innovation Guelph’s Agri-Innovation Accelerator (Innovation Guelph). Once our minimum viable product has been established and we reach a Technology Readiness Level (TRL) of 4 onwards, we would apply for accelerators like Bioenterprise (TRL 6 companies) and agriNEXT by Foresight Canada (TRL 4-9+) (Foresight). Once our startup becomes strong through these two stages of activities, we will seek investor opportunities that focus on climate action and agriculture, such as Pangea Ventures, Renewal Funds, and Cycle Capital Management. These were chosen from a list of investors outlined by the Canadian Venture Capital & Private Equity Association, referenced to us at SynBio Canada 2022 for our search (CVCA). The voiced offer for help from Bioenterprise already puts us at an advantage to tailoring our applications for success in funding through building a significant network and community.</p>
<p>The strong exporting infrastructure around wheat in Canada mentioned in section 2.3 will help our export into international streams, which will in turn increase international value and reach that will support developing operations in other countries. <b>Canada’s deregulation of wheat exports makes it an attractive country to import wheat from, making our expansion attractive and viable in multiple countries</b> (AGMRC, 2022). Especially given our identified potential customers from multiple countries, it would be most impactful to eventually provide this technology to multiple countries.</p>
<p>Our analysis of future expansion in terms of international export takes into account countries that have trade agreements with Canada, those that have non-negligible wheat production, and countries that are not already consistent purchasers of wheat from another country. Over 75% of Canada’s trade comes from countries it has trade agreements with (Wikipedia), therefore the countries that have FTAs became our starting list. More exclusion criteria and their justifications are summarized in Table 1. Applying inclusion and exclusion criteria <b>generated a list of 10 countries that are most suitable to export to based on our inclusion criteria and their need for a strong source of wheat (see Table 2), as well as the EU and UK and their countries</b>. The list includes Israel, Chile, Peru, Colombia, Jordan, Australia, Japan, New Zealand, Singapore, Vietnam, and countries in the EU and UK, which need to be expanded and further analyzed.From this list, a closer look at the impact of heat on their markets will be applied to come up with a shorter initial list. Countries that are in the top 10 wheat producers could also be a good market to export into, but these would require further analysis to ensure that they would have a large enough need for heat-resistant wheat specifically, therefore are included in the exclusion criteria in this initial screen.</p>
<br><br>
<table>
<tr>
<td><b>Inclusion Criteria</b></td>
<td><b>Exclusion Criteria</b></td>
</tr>
<tr>
<td>
Is a country with a trade agreement with Canada
<ul>
<li>Most of Canada’s exports (~75%) come from countries it has free trade agreements with. Without a trade agreement, strategies such as hiring an export intermediary like an export trading company (ETC) would have to be introduced, increasing costs and effort (Export.gov)</li>
</ul>
</td>
<td>Not a top 10 producer of wheat in the world</td>
</tr>
<tr>
<td>
Is not a country with a trade agreement with Canada
<ul>
<li>These countries would be self-sustained and would not need an additional source of wheat seeds. Even if their yield would decrease due to heat, generating a need or desire for alternative seed, a larger production operation means that there is a higher chance of offsetting these losses due to the size of backup operations</li>
</ul>
</td>
<td>Top 10 wheat producers in the world</td>
</tr>
<tr>
<td>
Does not have a strong wheat trade relationship with another country (<80% of their wheat comes from a singular other country)
<ul>
<li>Countries that rely heavily on others for their wheat imports would not easily switch a strong exporting relationship to one with a Canadian company. Even if a country like this would have yield loss due to heat, the effects would affect the industry less since the majority of their wheat comes from imports from another country</li>
</ul>
</td>
<td>Has a strong wheat trade relationship with another country (>80% of their wheat comes from a singular country)</td>
</tr>
<tr>
<td>
Have a significant wheat market
<ul>
<li>Countries that have small wheat production industries might not want to invest in a costly alternative for a market that is a small share of their production operations</li>
</ul>
</td>
<td>Wheat market is extremely small or nonexistent due to lack of land to grow grain</td>
</tr>
<caption>Inclusion and exclusion criteria for choice of countries to export to</caption>
</table>
<br><br>
<table>
<tr>
<td><b>Country</b></td>
<td><b>Meets inclusion or exclusion criteria?</b></td>
<td><b>Reason for exclusion</b></td>
</tr>
<tr>
<td>United States of America</td>
<td>Exclusion</td>
<td>Top 10 wheat producers</td>
</tr>
<tr>
<td>Mexico</td>
<td>Exclusion</td>
<td>Strong trade relationship with USA (FAS 2020)</td>
</tr>
<tr>
<td>Israel</td>
<td>Inclusion</td>
<td>90,000 tonnes per year (knoema)</td>
</tr>
<tr>Chile</tr>
<tr>
<td>Costa Rica</td>
<td>Exclusion</td>
<td>Land is unsuitable for wheat farming</td>
</tr>
<tr>
<td>Peru</td>
<td></td>
<td></td>
</tr>
<tr>
<td>Colombia</td>
<td></td>
<td></td>
</tr>
<tr>
<td>Jordan</td>
<td></td>
<td></td>
</tr>
<tr>
<td>Panama</td>
<td>Exclusion</td>
<td>Land is unsuitable for wheat farming</td>
</tr>
<tr>
<td>Honduras</td>
<td>Exclusion</td>
<td>Land is unsuitable for wheat farming (International Trade Administration)</td>
</tr>
<tr>
<td>South Korea</td>
<td>Exclusion</td>
<td>Strong trade relationship with US (FAS 2020)</td>
</tr>
<tr>
<td>Ukraine</td>
<td>Exclusion</td>
<td>Top 10 wheat producers</td>
</tr>
<tr>
<td>EU</td>
<td></td>
<td></td>
</tr>
<tr>
<td>Australia</td>
<td>Inclusion</td>
<td>25 million tonne per year (Aegic)</td>
</tr>
<tr>
<td>Brunei</td>
<td>Exclusion</td>
<td>Does not have wheat production</td>
</tr>
<tr>
<td>Japan</td>
<td>Inclusion</td>
<td>4.9 million tonnes per year (APH)</td>
</tr>
<tr>
<td>New Zealand</td>
<td>Inclusion</td>
<td>400,000 tonnes per year (Ravensdown)</td>
</tr>
<tr>
<td>Signapore</td>
<td></td>
<td></td>
</tr>
<tr>
<td>Vietnam</td>
<td></td>
<td></td>
</tr>
<tr>
<td>United Kingdom</td>
<td></td>
<td></td>
</tr>
<caption>Countries that Canada has Free Trade Agreements (FTA) with, with inclusion and exclusion applied to them to generate final included countries in the export plan.</caption>
</table>
<br><br>
<h3>Operations Expansion</h3>
<hr>
<p>As mentioned, the strength of the top 4 wheat producers that are not Canada (China, India, Russia, and the United States) offers a poor outcome in terms of exporting our product to them, but <b>offers an opportunity in establishing a production operation in these countries that can be inserted into their strong wheat production stream to maximize operational yield</b>. Recent heat waves in India have specifically affected crop yield to the point where valuable exports had to be halted in order to meet domestic demand (CNN). In combination with the fact that they are the second largest consumer of wheat in the world (IndexMundi), it is clear that <b>India’s extremely strong production stream is severely affected by heat, making them an attractive potential buyer of our product</b>. Wheat yield has decreased by 4.4-10% in 2022 due to heat waves, and our system can aid yield recovery from the temperatures that India has been suffering. We propose that introduction of our innovation can recover a modest percentage of India’s recently lost wheat yield in 2022, bringing the approximately 10% projected loss in yield to 100 million tonnes back up.</p>
<p>India is also among the countries that faces the largest inequality in economic profit between small-scale and large-scale farmers (FAO, 2021). Small-scale farmers have a decreased ability to jump back from crop yield loss from events like heat due to limited resources and technologies to restart planting and harvesting operations. As such, we expect that our technology has an increased benefit to small-scale farmers since it will be able to prevent such events, resulting in increased productivity and profit. Since our technology will aid to mend this gap between small-scale and large-scale production, <b>we expect the large small-scale farming community of India to take an appeal to our product, increasing the success of this operations expansion</b>.</p>
<br><br>
<h2id="LongTermImpact">Long-Term Impact</h2>
<hr>
<h3>Expected Positive Impacts</h3>
<hr>
<p>In the long term, we expect that our project’s outcomes will benefit the global community by helping the efforts towards addressing the sustainable development goals. The way our project is contributing to the sustainable development goals of Zero Hunger, Sustainable Production and Consumption, and Climate Action is outlined in our SDG Page. The content about positive impacts of our project in the SDGs page is written with the Entrepreneurship page’s outcomes in mind, so consider it as our writing in this section about our project’s positive impacts.</p>
<p>Apart from the positive global and social impact created by contributing to the sustainable development goals, we expect our project to positively impact farmers economically. Many farmers usually have yearly profit loss due to crop loss in high temperatures. Reducing the amount of crop loss by using heat resistant seeds will result in more profit. We expect this to specifically positively affect small-scale farmers since larger scale farmers will be able to recover from losses faster and with fewer overall negative effects. If our product successfully does reduce crop loss, it would improve the outlook on GMOs and their utility for society, reducing misconceptions around them.</p>
<br><br>
<h3>Expected Negative Impacts</h3>
<hr>
<h4>Ecological negative impacts</h4>
<p>A GMO usually introduces the risk of outcompeting native species if it exists without containment. However, since food crops like wheat are introduced by humans into a moderately controlled agricultural environment, this risk is low in the system it is affecting. There is still a possibility that it could eliminate native species if not properly contained and released into the environment, causing ecological imbalance. To manage this risk, we are planning on adhering closely to the confined field trial guidelines on preventing bio-contamination between GM plants when testing for approval from Health Canada, not only in the field trial but as a guideline for future farm users as well. The current mitigation includes keeping a distance of 2 meters between wheat and other plant species when growing and harvesting, as well as keeping the growing area free of other crops after the wheat season is gone. We know that crop rotation is an important part of encouraging soil health, therefore we recommend rotation from our modified spring wheat during the summer months to winter wheat during the winter months to maintain the soil health benefits of crop rotation while preventing the introduction of our genes to crops other than wheat.</p>
<br><br>
<h4>Human-based negative impacts</h4>
<p>The plasmids we are currently testing in protoplasts contain antibiotic resistance genes in order to be cloned through antibiotic selection in E. coli. Once stably integrated into whole wheat genomes, the existence of these genes in our plasmids could mean the accidental transfer of the genes to bacteria growing on the wheat through horizontal gene transfer, reducing the effectiveness of medicines. Even though horizontal gene transfer from plants to bacteria is relatively rare, it does occur (Nielsen et al. 2006); this gene transfer could also lead to gene alterations to other organisms. Additionally, the introduction of novel genes to the wheat genome could inadvertently alter other genetic pathways, potentially triggering allergic responses by creating plant allergens that did not previously exist in the plant (Oak Ridge).</p>
<br><br>
<h4>Economic negative impacts</h4>
<p>Before the scale-up process described above, our technology is likely to be unequally accessible to different populations due to its increased price as laid out in the cost analysis. As a result, small-scale farmers may suffer from the introduction of our technology from medium and large-scale farmers outcompeting their productivity even more than usual due to their increased yield with the introduction of our technology.(EEA). To manage this risk, our cost analysis includes a cost reduction plan,to prevent harming the small-scale farmers we are aiming to benefit and target. We recognize that this disparity between the desire to target small-scale farmers, but the technology being least accessible to them in initial years, is a limitation of our commercialization plan to overcome through cost reduction and proper distribution planning.</p>
<br><br>
<h4>Social negative impacts</h4>
<p>Lack of regulation and information about using GM foods in developing countries could result in misuse of our technology, spreading misinformation about the benefits and risks of genetically modified foods and resulting in less access to this technology in regions that require it the most (Oye et al. 2014). To manage this risk, we have conducted two panels educating the public on the benefits of genetically modified organisms, and to discover the disparity in synthetic biology innovations between developing and developed countries. As described in the Outreach wiki page, we hope that these panels were able to increase acceptance of GMOs in order to reduce the potential misuse or misunderstanding of our technology, and identify how developing countries can increase infrastructure and acceptance to GMOs by taking examples from other countries, respectively. We hope that the result will be to impact people who can similarly educate the target audience for our technology, such as small-scale farmers in multiple regions of the world.</p>
<br><br>
<h3>How to Measure Project Impact</h3>
<hr>
<p>In reflecting on our project implementation and commercialization, we find it valuable to not only list our expected positive and negative impacts, but also mention how we will measure this impact. For example, while 62% of companies in 2017 mentioned contributing to an SDG, only 28% set quantitative targets to measure their impact, allowing for lack of accountability (Sopact). We envision we will measure how closely we are reaching these positive and negative impacts through measuring the social return on investment (SROI), which has been integrated into our development timeline. SROI accounts for social and environmental value of innovations through assigning a monetary value to a company’s activities (Sopact 2). This generates a SROI Ratio which indicates how many dollars worth of social value is outputted from every dollar allocated to the company. It can be both predictive (in our case can be implemented during pre-development) and evaluative, which can be implemented after commercialization.</p>
