<p>For the realization of our product, we developed a commercialization strategy, through which we could make the transition from the lab to the market. In this section, we have outlined steps for the commercialization of our product.</p>
<li><strong>Research and development:</strong> In this stage, we will continue refining our technology as a start-up, ensuring it is scalable, reliable, and can be applied not only to eukaryotic model organisms, but also to a range of crops and agricultural systems. Following this, we will conduct pilot studies and field trials with focus groups of key stakeholders (e.g. innovative farmers or research institutions like TU Delft AgTech Institute, which is already a sponsor of our project) to validate the effectiveness of nitrogen-fixing crops in real-world conditions. Our final challenge in this phase would then be to navigate regulatory hurdles, such as getting approval from agricultural and environmental agencies. We would hire an expert to guide us through this (more on this in the skill gap analysis). Along with the hiring, we will ensure that our product complies with biosafety standards and obtain certifications needed for commercial agriculture using these seeds in The Netherlands, for example the seed certification of the Netherlands General Inspection Service for Agricultural Seeds and Seed Potatoes (NAK), which ensures that GM seeds meet standards related to safety, quality, and environmental protection.</li>
<li><strong>Intellectual property protection:</strong> In this phase, we will aim at securing strong intellectual property protection for our technology, including patenting our unique genetic engineering methods or products such as the fusion with which we incorporate the Nitroplast in a cell, as well as the resulting seed itself. For these steps, we will also make use of our skill gap analysis and hire the corresponding experts on intellectual property law. Following this, we will refine and protect the brand identity of our technology through trademark registration, which is the legal process of registering a specific symbol, name, or logo to obtain exclusive rights to use it. This can be done for any other distinctive signs that identify and distinguish our product.</li>
...
...
@@ -79,7 +79,7 @@
<li><strong>Revenue model:</strong> Our revenue model would first focus on the direct sale of the seeds to farmers and farmers’ collectives. Once we have licensed our technology to large seed producers, we would also acquire a share of the profits made by them via NitroBLAST seeds.</li>
<li><strong>Scaling and growth:</strong> At first, we would start by selling to the Dutch market. This is because although the nitrogen crisis is a global issue, its effect is particularly pronounced in the Dutch landscape. Following this, we would leverage partnerships with global agricultural companies and NGOs to expand into new territories and collaborate with global sustainability initiatives to align with regional regulations and goals. This includes government subsidy programs for sustainable agriculture that will further aid in reducing the financial burden on farmers.</li>
</ol>
</ul>
<divclass="h2">Lean Canvas Business Model</div>
<p>The Lean Canvas is a simplified, one-page business model framework designed to help entrepreneurs and startups quickly outline and validate their business ideas. The focus is on identifying key assumptions, problems, and solutions early on, allowing for faster testing and iteration. We have designed the following Lean Canvas for our business:</p>
<li><strong>Disrupting the Fertilizer Market:</strong> Our product has the potential to disrupt the fertilizer market by reducing, or even eliminating, dependence on synthetic fertilizers. With the later increase in regulations and taxes on nitrogen fertilizers, our technology is an attractive alternative for those who want to save some money while still getting optimized results.</li>
<li><strong>Environmental Policy Alignment:</strong> Governments and international organizations are increasingly pushing for sustainable agricultural practices. Our seeds align with these policies, which could make our company eligible for subsidies and other incentives.</li>
...
...
@@ -160,7 +160,7 @@
<li><strong>Potential Partnerships:</strong> Partnering with big agricultural companies could rapidly increase our market share and credibility. Additionally, by partnering with environmental organizations, such as WWF or Greenpeace, we could promote our product as a new sustainable farming practice, fighting against the previously mentioned negative public perception of GMOs.</li>
<li><strong>Technological Evolution:</strong> As the technology evolves, we could incorporate other beneficial traits into the plants that could also improve the harvest and facilitate the farmers' work, such as making them drought-resistant, pest-resistant, etc.</li>
<p>The TAM represents the global agricultural industry's full potential for our solution. This includes all farmers globally who use synthetic fertilizers, across all crop types. Globally, the fertilizer market is valued at over 200 billion USD annually, with nitrogen-based fertilizers comprising a significant portion of that. Since almost all major crops (wheat, rice, maize, etc) depend on nitrogen inputs, the TAM includes every agricultural sector that relies on fertilizer.</p>
<divclass="h2">Serviceable Available Market: SAM</div>
<p>The SAM represents the portion of the TAM that our solution could realistically serve, based on the specific crops and regions where its application could be useful. The SAM would direct our focus to regions with severe nitrogen pollution issues, such as Europe, North America and parts of Asia. In these regions large scale farms producing cereals like maize, and other staple crops would be our primary targets. The European nitrogen fertilizer market alone is valued at approximately 50 billion USD and the US market is another 15 billion USD. Including other developed nations and regions with high agricultural output, the SAM could easily represent 80-100 billion USD in potential revenue. Key crops like wheat, maize, rice, and soybeans—staples that require heavy nitrogen inputs—would represent the initial market for our solution.</p>
<p>The SOM is the realistic segment of the SAM that we could capture within the first few years of commercialization, considering competition, market penetration strategy, and resources. The initial target could be innovative and sustainability-focused farmers in developed regions, specifically those facing strict environmental regulations like the Netherlands, Germany, and parts of the U.S. Focusing on early adopters—perhaps 2-5\% of the SAM in high-regulation regions—our initial obtainable market could be worth 2-5 billion USD. We would then start with the Netherlands, given the severe nitrogen crisis, and expand into other parts of Europe and North America where government incentives and environmental policies favor sustainable solutions.</p>
<p>A skill gap analysis identifies the skills and knowledge that our current team lacks that are crucial for achieving our objectives. Our team already has solid technical knowledge in areas like nanobiology, maths, physics, life science, technology, software design, and biomedical engineering, all of which are important for developing the product. However, there are still gaps in some other areas critical for the correct development of the product.</p>
<p>Even though we have a solid background in related fields, the development of a GMO with new organelles requires more specialized knowledge in genetic engineering, including technologies such as CRISPR, and also in plant biotechnology. We would need experts in manipulating plant genomes to integrate nitroplasts effectively and ensure the plant’s overall correct growth. Within this line, we would also need plant physiology experts to make sure that the nitroplasts will not negatively affect other functions of the plant and agronomists to determine how modified seeds will perform in different environmental and soil conditions.</p>
<p>Given the nature of our product, taking into consideration the current laws and regulatory environment in different countries is critical. For this, we would need a specialist in GMO regulatory affairs: a specialist in GMO laws and regulations in different countries, including environmental risk assessments and safety standards. We would also need expertise in patent law, especially in plant patents, who can guide us in intellectual property for genetic modifications. Finally, we would also need an expert in environmental law to manage concerns about the ecological impact of introducing a GM plant that fixes nitrogen and to conduct an environmental impact assessment.</p>
<p>To commercialize our product effectively, we need expertise in agro-business strategy and market analysis. Specialists in this area would help us understand market demand, identify customer segments such as farmers or agricultural companies, and navigate competitive dynamics in the agriculture sector. Additionally, we need expertise in supply chain management to handle the logistics of producing and distributing the seeds while ensuring their quality and compliance with regulations.</p>
<p>Financial expertise specific to biotechnology and agro-tech is also critical. Developing GM crops requires significant capital investment, and financial professionals can help with financial modeling, securing investment, and managing investor relations. This will ensure that we have the necessary funding and a sound financial strategy to support our product development and market expansion.</p>
<p>To ensure smooth integration of our seeds into farming practices, we would need agricultural engineers who understand how to modify or develop farming machinery for planting and harvesting our crops. We also need experts in farmer education and agricultural extension services to help guide farmers in adopting the new seeds and integrating them effectively into their existing agricultural systems. This would be essential for maximizing crop benefits and ensuring broad adoption of our product.</p>
<p>Last but not least, addressing ethical, social, and environmental considerations is equally important. A bioethicist will help us navigate public concerns about GMOs, ensuring that our communication strategy is sensitive to ethical questions around food safety and biodiversity. Finally, we would also require sustainability and ecological impact specialists to assess the possible long-term effects of our nitrogen-fixing crops and align our product with global sustainability goals, ensuring that it is both environmentally responsible and marketable.</p>
<p>An exit strategy is a planned approach by which the owners or investors of a company aim to sell their stake in the business, either fully or partially, to achieve a return on their investment. Genetically modified organisms require a significant initial investment, as substantial funding is necessary for the research and development of such products. Therefore, it is crucial to thoroughly assess the potential for profit to ensure that it is, indeed, a sound investment.</p>
<p>One of the most common exit strategies involves allowing a large company, in this case specialized in biotechnology or the agricultural sector, to acquire our small business. Technology is advancing rapidly, and large companies cannot afford to fall behind. Acquiring our company, and thus our ideas, would allow them to stay current and maintain their market leadership. To attract potential buyers, we need strong intellectual property and patenting, as well as a solid proof of concept, to ensure profitability for these larger companies.</p>
<p>In a similar vein, but without losing our personal identity, forming partnerships will be essential for a successful market strategy. These partnerships could be with companies that complement us in technology, resources, or that already have a recognized position in the market and established contacts. This would create value for both companies and allow us to reduce costs and accelerate market entry, enhancing our overall competitiveness in the agricultural sector.</p>
<p>Another option would be to take our company public through an initial public offering (IPO). An IPO is a public offering in which shares of a company are sold to investors. In this way, although the ownership of our company would be shared with investors, we could raise substantial capital. To attract investors, we would need to demonstrate clear revenue growth and engage financial advisors to guide us through the process.</p>
<p>Finally, another possible exit strategy, inspired by one of our Human Practices interviews, involves licensing our technology to other companies. By doing this, we could generate revenue through royalties while maintaining ownership of our intellectual property. For this, we would again need to secure a patent and design various licensing models that could attract potential buyers. Our originality would be the only limit to these packages.</p>
<p>Even though all these ideas could have great results, we have decided to opt specifically for the last one, to have an alignment with our Human Practices team.</p>
<p>One promising approach to balance the need for fertilizer and the welfare of the environment, is the development of plants that can fix atmospheric nitrogen independently. This innovation would not only reduce the need for synthetic fertilizers and manure but also help mitigate climate change and the nitrogen crisis. <strong>To this end, we first need to better study the nitroplast, how it interacts with the host organism and how it could be potentially introduced into other cells</strong>.</p>
<p>It has been discovered that, to ensure the endosymbiotic relationship, several proteins that are essential to UCYN-A are expressed in the host, <em>B. bigelowii</em>, and imported into the symbiont, similar to chloroplasts and mitochondria, though to a lesser extent <ahref="#cite11"style="color: #185A4F;">[11]</a>. Many of these proteins possess specialized localization peptides that direct their cellular function. In UCYN-A, these peptides are usually a C-terminal extension and are known as the “uTP” (UCYN-A Transit Peptide), although not yet identified <ahref="#cite11"style="color: #185A4F;">[11]</a>. Our first aim was to employ bioinformatics analyses to identify the characteristic <strong>motifs required for a protein to be imported by UCYN-A</strong>. For this, we made use of host (<em>B. bigelowii</em>) and nitroplast (UCYN-A) genome data as well as the proteomics data published by Coale <em>et al.</em>. <strong>We identified 2 putative uTP sequences with high likelihood, which we named uTP1 and uTP2</strong>.</p>
<p>To understand the functioning of the UCYN-A import mechanism, we attempted to <strong>identify the proteins involved in translocating</strong> host-encoded proteins into UCYN-A. First, we located genes in the host genome that are potentially involved in the translocation, based on their similarity to proteins in other import mechanisms such as from <em>Paulinella chromatophora</em> (UCYN-A analogue for photosynthesis). Potential chaperones analogous to heat-shock proteins were also included in the search. These chaperones are hypothesized to bind to proteins tagged by the uTP and keep them from folding, allowing translocation through the UCYN-A membrane. We then followed this by <strong>obtaining the tertiary structure of all candidate proteins</strong> using a structure prediction tool, and used <strong>docking</strong> tools to select candidate proteins likely to bind the previously identified transit motifs.</p>
<p>In addition to <em>in silico</em> experiments, we also aimed to investigate the transport mechanisms of UCYN-A <em>in vivo</em>. Instead of making use of plants as target organisms, we opted for using single-cell model eukaryote organisms, namely the yeast <em>S. cerevisiae</em> and the green alga <em>C. reinhardtii</em>. The initial <em>in vivo</em> characterization of the UCYN-A transport system involved <strong>examining the expression and localization of the UCYN-A transit peptides in these eukaryotic model organisms</strong> to test whether uTP would have any unexpected effect on cell viability and would not target any other organelle. To this end, we designed vectors, cloned them using Gibson-assembly, transformed bacteria, purified expression plasmids, and transformed those into <em>S. cerevisiae</em> and <em>C. reinhardtii</em>. We expressed uTP-tagged fluorescent proteins, together with controls targeting other organelles, and localization was assessed using fluorescence microscopy. Our preliminary results indicate that uTP did not target any other organelle and did not lead to alterations in cell morphology.</p>
<p>Studies have demonstrated the insertion of bacteria into cells by engineering endosymbionts in <em>S. cerevisiae</em> using either <em>E. coli</em> or <em>S. elongatus</em><ahref="#cite12"style="color: #185A4F;">[12]</a>. Another study successfully inserted <em>Azotobacter</em> strains into <em>C. reinhardtii</em><ahref="#cite13"style="color: #185A4F;">[13]</a>. Building on this research, we initially aimed to <strong>develop a reliable protocol for transplanting a nitroplast</strong> into <em>C. reinhardtii</em> and <em>S. cerevisiae</em> as a <strong>proof-of-concept</strong> for transplantation into other eukaryotes, using polyethylene glycol (PEG) fusion protocols. However, due to time limitations, we started out with the model eukaryotic bacteria, <em>E. coli</em>, and refined a protocol for its fusion with <em>S. cerevisiae</em>.</p>
<p>We obtained <em>B. bigelowii</em> as a gift from Dr. Kyoko Hagino (Kochi University, Japan) and cultured it according to the protocol. For the transplantation of the nitroplast into a different model organism, we were also required to isolate this organelle from its host. Our project also implemented the protocol for nitroplast isolation from its host, according to the protocol and suggestions provided by Dr. Tyler Coale (University of California, San Diego, USA). Isolated UCYN-A could potentially be used for PEG fusion with other eukaryotes.</p>
<p>Finally, we have also wondered how our project would affect society. For this, our human practices team has actively worked to understand the possible consequences of our project. Also, wider acceptance of our idea goes hand in hand with educating the general population. Last but not least, we have also assessed the potential economic benefits of our idea and the omission of fertilizer use in agriculture. For this, we made an <strong>economic analysis</strong> and business plan.</p>
<p>Our project <strong>lays the foundation for the transplantation of nitroplast</strong> into eukaryotic hosts. The emergence of nitrogen-fixing plants could lead to a significant drop in the demand for fertilizers, and consequently in both carbon emissions and nitrogen pollution.</p>
</div>
<p>Since our project is still in the R&D phase, our team has not filed for a patent yet. However, as our research continues and we establish the final proof-of-concept of our technology, we aim to patent not only the technology used to produce the seeds but also the seeds themselves, allowing us to license these seeds to other seed companies. Ultimately, since our goal is to reduce nitrogen emissions and contribute to the environment, we plan to make these seeds available to countries facing persistent food security issues through NGOs and other social service organizations.</p>
</div>
<!-- 3 -->
<divclass="h"id="six">
<divclass="h1">Long Term Impact</div>
<p>Although the long-term impact of GMOs, both environmentally and on consumers, is unknown, we can attempt to estimate the economic impact if our product were to succeed and be accepted by the public.</p>
<divclass="h2">Would We Truly Eliminate the Need for Fertilizers?</div>
<p>
To determine whether our potential crops could be self-sufficient, we calculated whether they could indeed fix the total amount of nitrogen they need for growth. We started by examining the nitrogen fixation rates of UCYN-A, using data from <spanclass="cite">CITE</span>. There are different nitrogen fixation rates for UCYN-A1 and UCYN-A2. The one that is an endosymbiont with <i>B. bigelowii</i> and represents an early stage of an organelle is UCYN-A2.
</p>
<p>
"We speculate that the <i>B. bigelowii</i> endosymbiont may represent an early stage of endosymbiosis before it is fully established as an organelle, and it disappears under ammonia-rich conditions, in contrast to UCYN-A1." <spanclass="cite">CITE</span>, which is why we used the fixation rate for UCYN-A2, which is 151.1±112.7 fmol/(cell·day) of nitrogen. Let's suppose that each cell contains one and only one nitroplast (this is, an UCYN-A2). The number of cells in a crop plant can vary from the kind of crop and other multiple factors, but it is a number between a billion up to a trillion cells. Let's say that the average plant has 50 billion cells, as an approximation, depending on the specific variety of crop and its growth conditions. Using this value, we get that our genetically modified plant would fix 0.1511 mmol per day. If we take into consideration diatomic nitrogen's molar mass, 28.02 g/mol, our plant would fix 211.65 mg/day. Plants need less than 100 mg per day to survive, so our plants would be completely self-sufficient.
</p>
<pre>
$$
\text{nº of cells in 1 plant} \times \text{nº UCYN-A2/cell} \times \text{UCYN-A2 fixation rate} \times \text{conversion rates} = 211.65 \text{ mg/day}
$$
</pre>
<divclass="h2">How Much Money Would We Save?</div>
<p>Being a team from the Netherlands, we have actively followed the <strong>unfolding of the nitrogen crisis</strong> and seen the farmer's protests on the news. While nitrogen deposition is incredibly harmful to the environment, the Dutch agriculture sector is a big driving factor behind its economy, with <strong>agricultural exports</strong> being worth 124 billion euros in 2023 alone <ahref="#cite8"style="color: #185A4F;">[8]</a>.</p>
<p>The Netherlands is also considered one of the <strong>front runners in terms of food and agriculture technology</strong>. Given the leadership of The Netherlands in this field, why not leverage synthetic biology to address the nitrogen crisis? We were inspired by previous iGEM teams such as Wageningen 2021 <ahref="#cite9"style="color: #185A4F;">[9]</a> and Stony-Brook 2023 <ahref="#cite10"style="color: #185A4F;">[10]</a> that have tackled similar challenges, alongside a recent publication in Nature in April 2024 <ahref="#cite11"style="color: #185A4F;">[11]</a>.</p>
The main goal of our solution is omitting the use of fertilizers. Therefore, firstly, the amount of money that we would save primarily depends on the operating costs of a farm that goes towards purchasing fertilizer. We made an analysis for this amount for the Netherlands as well as the top 4 agricultural producers of the world, China, USA, Brazil, and India. We made this analysis for the most nitrogen-utilizing crops: sugar beet, maize, wheat, and rapeseed. Since Brazil and India do not intensively produce sugar beet but do produce sugar cane, we also made this calculation for these countries. In this analysis, we made a few assumptions. The first assumption was that the nitrogen-based fertilizer being used was urea-based, which has 46% nitrogen. The second assumption was that the use of NitroBLAST seeds would not increase the operational costs of the farm significantly, as compared to the use of regular seeds. The calculation was then done in the following way. For example, for the cultivation of maize in China, annually 8.75 million tonnes of urea-based fertilizer is applied. The cost of this fertilizer is 2.54 USD per metric ton, leading to an annual estimate of 3.44 billion USD spent in China on urea-based fertilizer for the nitrogen supply required for the cultivation of maize. You can see the analysis done for the other crops and countries in the following plot: <i>insert plot here</i>.
</p>
<p>
The amount of money that we would save would also depend on the efficiency of our product. We say that our product is 100% efficient if the grown plant can fixate up to 211.65 mg of nitrogen per day. We will now do an estimation of how much money we would save as a function of the efficiency of our product. To make this as accurate as possible, we are going to focus on one crop type and country. As we are a Dutch team, we will focus on the Netherlands, and for crop we will go for maize, as it is one of the most popular ones: last year, the Netherlands exported 2,621,490 kg of maize<spanclass="cite">CITE</span>. In total, there are around 200,000 hectares of maize throughout the country<spanclass="cite">CITE</span>. The typical application rate is around 155 kg of nitrogen per hectare (as recommended by WUR), which would mean that approximately 31 million kilograms of pure nitrogen are applied. Depending on which kind of fertilizer is used, this would be one amount of fertilizer or another. We will assume that we are working with urea-based fertilizer, as it is one of the most used in the Netherlands. The nitrogen content of urea-based fertilizer is 46%. This means that, in total, 67.4 million kilograms of urea fertilizer would be applied; this is, 67,400 tons of fertilizers. The price of urea fertilizer is 350 euros per ton. This would mean that, if we follow our assumption of urea fertilizer use, the Netherlands would be spending 23.6 million euros only on fertilizer for maize.
</p>
<pre>
$$
\text{nº ha of maize} \times \text{N}_2 \text{ application rate} \times \text{conversion to urea fertilizer} \times \text{price of fertilizer} = 23.6 \text{M euros}
$$
</pre>
<p>
We can also compute how many milligrams of nitrogen a maize plant needs per day. We already know that the general nitrogen requirement per hectare is 155 kg. As the typical plant density is 85,000 plants per hectare, and if we also consider that the typical maize growing season spans about 120 days, we get that a single maize plant in the Netherlands requires approximately 15.2 milligrams of nitrogen per day on average, as opposed to the 211.65 mg that it would be theoretically able to fix.
This would mean that we would only need a 7.18% efficiency to cover 100% of the plant's needs. As we can see in the following graph, even a small efficiency can lead to remarkable savings.
<p>Being a team from the Netherlands, we have actively followed the <strong>unfolding of the nitrogen crisis</strong> and seen the farmer's protests on the news. While nitrogen deposition is incredibly harmful to the environment, the Dutch agriculture sector is a big driving factor behind its economy, with <strong>agricultural exports</strong> being worth 124 billion euros in 2023 alone <ahref="#cite8"style="color: #185A4F;">[8]</a>.</p>
<p>The Netherlands is also considered one of the <strong>front runners in terms of food and agriculture technology</strong>. Given the leadership of The Netherlands in this field, why not leverage synthetic biology to address the nitrogen crisis? We were inspired by previous iGEM teams such as Wageningen 2021 <ahref="#cite9"style="color: #185A4F;">[9]</a> and Stony-Brook 2023 <ahref="#cite10"style="color: #185A4F;">[10]</a> that have tackled similar challenges, alongside a recent publication in Nature in April 2024 <ahref="#cite11"style="color: #185A4F;">[11]</a>.</p>
