<p>UCYN-A is actively undergoing genome reduction as part of its evolution towards an organelle: the reason it cannot live independently is that essential proteins for its survival are no longer present in its genome, but are now encoded in B. bigelowii’s and then imported into UCYN-A.
Using recently released proteomics data on B. bigelowii and UCYN-A, along with older genomics and transcriptomics data, we have identified a putative list of proteins that are imported into the organelle. This data provides a solid foundation for further research into which proteins are essential, as we suspect many are redundant. Identifying a list of essential host-encoded proteins is crucial to successfully transplanting UCYN-A into a new host.
</p><p>We have also created a new transcriptome assembly of B. bigelowii based on raw data from previous studies, using improved algorithms. This allowed us to create a new predicted proteome.
We are making all of our omics data available for future iGEM teams along with documentation. </p>
<p>You can read more <ahref="https://2024.igem.wiki/tu-delft/results">here</a>
<p>UCYN-A is actively undergoing genome reduction as part of its evolution towards an organelle: the reason it cannot live independently is that essential proteins for its survival are no longer present in its genome, but are now encoded in <em>B. bigelowii</em>’s and then imported into UCYN-A.
Using recently released proteomics data on <em>B. bigelowii</em> and UCYN-A, along with older genomics and transcriptomics data, we have identified a putative list of proteins that are imported into the organelle. This data provides a solid foundation for further research into which proteins are essential, as we suspect many are redundant. Identifying a list of essential host-encoded proteins is crucial to successfully transplanting UCYN-A into a new host.
</p><p>We have also created a new transcriptome assembly of <em>B. bigelowii</em> based on raw data from previous studies, using improved algorithms. This allowed us to create a new predicted proteome.
We are making all of our omics data available on request for future iGEM teams along with documentation. </p>
<p>You can read more <ahref="https://2024.igem.wiki/tu-delft/results">here</a>.
</p>
</div>
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<divclass="h1">UCYN-A transit peptide</div>
<p>Based on previous studies and the proteomics data on B. bigelowii we identified a strongly preserved motif on the C-terminal end of B. bigelowii proteins that are imported to UCYN-A. This sequence was hypothesized to correspond to a transit peptide (UCYN-A transit peptide or uTP), responsible for localizing proteins to the organelle.</p>
<p>The characterization of the transit peptide is a crucial step in understanding the nitroplast protein import pathway in B. bigelowii. The part allows future teams to run experiments aiming to deliver proteins to UCYN-A, whether in its native host to investigate its behavior or within a different recipient cell, to create an environment where the nitroplast could survive if transplanted. This all has implications on advancing research toward engineering new nitroplast-containing, nitrogen-fixing eukaryote strains.</p>
<p>You can read more <ahref="https://2024.igem.wiki/tu-delft/results">here</a></p>
<p>Based on previous studies and the proteomics data on <em>B. bigelowii</em> we identified a strongly preserved motif on the C-terminal end of <em>B. bigelowii</em> proteins that are imported to UCYN-A. This sequence was hypothesized to correspond to a transit peptide (UCYN-A transit peptide or uTP), responsible for localizing proteins to the organelle.</p>
<p>The characterization of the transit peptide is a crucial step in understanding the nitroplast protein import pathway in <em>B. bigelowii</em>. The part allows future teams to run experiments aiming to deliver proteins to UCYN-A, whether in its native host to investigate its behavior or within a different recipient cell, to create an environment where the nitroplast could survive if transplanted. This all has implications on advancing research towards engineering new nitroplast-containing, nitrogen-fixing eukaryote strains.</p>
<p>You can read more <ahref="https://2024.igem.wiki/tu-delft/results">here</a>.</p>
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<divclass="h1">Peptide binding model</div>
<p>Based on the previously identified transit peptide and the predicted B. bigelowii proteome, we attempted to find any proteins which interact with uTP and thus might play a role in the UCYN-A protein import system. To achieve this, we began developing a proteome-scale peptide-binding prediction tool, which, to our knowledge, does not yet exist.</p>
<p>Based on the previously identified transit peptide and the predicted <em>B. bigelowii</em> proteome, we attempted to find any proteins which interact with uTP and thus might play a role in the UCYN-A protein import system. To achieve this, we began developing a proteome-scale peptide-binding prediction tool, which, to our knowledge, does not yet exist.</p>
<p>Unfortunately, due to the strict time constraints in iGEM, we were not able to finish work on this tool, but we are making our existing code and data available to aid any future teams.</p>
<p>Read more on our Materials and Methods page</p>
<p>Read more on our <ahref="experiments"style="color: #185A4F;">Materials and Methods</a> page.</p>
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<divclass="h"id="six">
<divclass="h1">Entrepreneurship dictionary</div>
<p>Over the course of our project, while we worked on our entrepreneurship case, we found a lot of entrepreneurship jargon. As students of a technical university, and early career starters, we were not familiar with a lot of these words. To make it easier for future iGEM teams working on entrepreneurship cases and potentially a start-up from their iGEM team, we compiled a list of words and their meanings relevant to entrepreneurship in an entrepreneurship dictionary. Through this we hope to help future iGEM teams that might encounter confusions similar to us.</p>
<divclass="h1"><em>B. bigelowii</em> on list A1</div>
<p>B. bigelowii is an organism of great interest thanks to UCYN-A. This is because the nitroplast is something like an “evolutionary snapshot” of organellogenesis, and its capability to fix nitrogen makes B. bigelowii the only known nitrogen-fixing eukaryote. However, B. bigelowii is not a model organism, and as a coccolithophore with no biosafety class assigned by any institution, it can prove complicated to gain authorization to work with it. Meanwhile, many experiments on our roadmap for the future of nitroplast transplantation involve modifying B. bigelowii, so this presented a problem.
<p><em>B. bigelowii</em> is an organism of great interest thanks to UCYN-A. This is because the nitroplast is something like an “evolutionary snapshot” of organellogenesis, and its capability to fix nitrogen makes <em>B. bigelowii</em> the only known nitrogen-fixing eukaryote. However, <em>B. bigelowii</em> is not a model organism, and as a coccolithophore with no biosafety class assigned by any institution, it can prove complicated to gain authorization to work with it. Meanwhile, many experiments on our roadmap for the future of nitroplast transplantation involve modifying <em>B. bigelowii</em>, so this presented a problem.
</p>
<p>We have worked together with our department's biosafety officer to submit a formal request to the Dutch National Institute for Public Health and the Environment (RIVM) for B. bigelowii to be approved into list A1. Organisms in this list are considered equal to common model organisms like E. coli in terms of biosafety and so they can be genetically modified in BSL-1 labs, insofar as no hazardous sequences are involved. We believe this classification will facilitate future research on B. bigelowii by Dutch teams immensely and should make it easier for teams from other countries to get clearance on experiments as well.</p>
<p>We have worked together with our department's biosafety officer to submit a formal request to the Dutch National Institute for Public Health and the Environment (RIVM) for <em>B. bigelowii</em> to be approved into list A1. Organisms in this list are considered equal to common model organisms like <em>E. coli</em> in terms of biosafety and so they can be genetically modified in BSL-1 labs, insofar as no hazardous sequences are involved. We believe this classification will facilitate future research on <em>B. bigelowii</em> by Dutch teams immensely and should make it easier for teams from other countries to get clearance on experiments as well.</p>
<p>We started by using the<strong> Value-Sensitive Design (VSD)</strong> approach because it helped us identify our project in relation to problems and values. It also facilitated a discourse between team members and stakeholders about the meaning of these values in our project and how to carry out a responsible approach. <ahref="#cite1"style="color: #185A4F;">[1]</a> This entailed thoroughly anticipating both positive and negative impacts of our project and thinking of safety and security and the ethical and social problems created by its potential application. </p>
<p>The steps of our <strong>HP</strong> and <strong>Integrated HP approach</strong>, based on the <strong>VSD analysis</strong>, are shown in <strong>Figure 1.</strong> The VSD consists of three main phases namely the conceptual, empirical and technical part. We applied these stages in our HP work. In the <strong> conceptual part</strong>, we assessed who are the stakeholders impacted by our idea and what values are at relevance. This way, we gained a better understanding of whom to engage with and what questions we wanted to ask. The <strong>empirical part</strong> consisted of reaching out to some of these stakeholders with different backgrounds, and to experts that could help us think about the different fields of impacts mentioned before. During the <strong>technical part</strong> we integrated all gathered information from the conceptual and empirical parts to minimise potential risks associated with our project and to come up with alternative approaches. This also meant that we had to make compromises between conflicting design choices. </p>
<figcaption>Figure 1: The map of our HP steps <br>The steps are the following: Identify the problem → Come up with solution and benefits→ Identify values and stakeholders→ Assess impact through talking to stakeholders: environmental, social and economic impact→ Implement: mitigate risks, think of alternative approaches </figcaption>
<p>As we learned from the conversations with different stakeholders, defining to what problem our idea serves as a solution is very important from the aspect of responsible innovation. It was also the first step of our VSD analysis. </p>
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<p>The identified/relevant values were food security, accessibility, social/environmental sustainability, safety. The value hierarchy of the two most important values safety and accessibility can be seen in Figure 4 and Figure 5 as an example.</p>
<p>Safety was found to be an important value for the European Union but also to the Dutch Government and the public. Safety can be divided into environmental and food safety. During our HP work we mostly dived deeper into the question of environmental safety related to our idea. Figure 3 shows how norms such as ‘No risk for the environment derives from the value safety and what are the certain design requirements such as ‘the genetically modified (GM) plant shouldn’t outcompete native species’ to satisfy those norms in our design. We later rediscussed these design requirements and modified them according to the information we gathered from interviews we conducted. Making design choices related to safety were difficult. The design requirements for safety often clashed with the ones derived from accessibility. This is discussed later.</p>
<p>Accessibility was an important value identified related to farmers and NGOs. NGOs like Greenpeace argue that the Agro and Seed industries main priority is profit (by patents and seeds that need to be rebought every year) rather than to make their technology and products accessible for all farmers and serve their local needs.[8] The design requirements shown in Figure 4 are interesting ones related to patenting and ownership, but also touching the core of our whole design. Other important questions for farmers are how expensive the GM seeds are. Is it affordable or cheaper compared to the non-GM type that needs fertilizer? Will the farmers have to buy the seeds every year? These questions related to accessibility touch the question of ownership and safety which are discussed in the IHP part.</p>
<p>See what design adjustments we made regarding these questions after interviews.</p>
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<p>We had the opportunity (with the kind help of TU Delft AgTech Institute) to have a critical discussion with four scientists from KWS SAAT SE & Co. KGaA about our idea and experimental approach. KWS is an international seed company. We thought it is relevant to talk about the feasibility of our idea and approach with experienced scientist from a company that is relevant to seed development.</p>
<p>During our talk with the scientists we discussed additional aspects that are important to test for our idea in the early phases. Therefore, we included additional experiments and approaches for the fusion experiments but also for characterising our uTP peptide. More details can be found on the Future wet-lab experiments page. Additionally, they raised their concerns about the feasibility of our idea. They highlighted that it is important to think of alternative approaches and how our idea could compliment already existing solutions for improving nitrogen-fixation in plants. Reflecting to this we discuss these possibilities under Alternative approaches. </p>
<p>Martijn Schaap is a Professor at Freie Universitaet Berlin on Air Quality and Principal Scientist at the Netherlands Organisation for Applied Scientific Research ( TNO ). TNO is an independent research organisation that aims to create innovations while collaborating closely with governments, universities and the private sector. [TNO website]</p>
<p>Since Martijn is an expert on reactive nitrogen emissions and deposition we could learn more about the situation in the Netherlands, what are the main sources of ammonia and nitrogen oxide emissions. Since he is a researcher at TNO which is a Dutch organisation, we learned how the Dutch government approaches the problem. He also gave his opinion on ideas that could help solve the problem, these are also discussed in the alternative approaches part.
<p>We wanted to implement the notion of responsible innovation during our project. That is why we contacted dr. Zoë Robaey who is currently an Assistant Professor in Ethics of Technology at the Philosophy Group of Wageningen University. Her work investigates moral responsibility under conditions of uncertainty in the field of biotechnology in agriculture.
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<p>We learned that it is not enough to have a potentially revolutionary idea that we think could do good. It is a fundamental part of being responsible that we think of how our idea or product will be used in society, who will own it, what exact problems our innovation will solve and what consequences can be anticipated to different choices. We developed our idea and thought of its application with this mindset all along.</p>
<p>As a result of our discussion, we came up with different types of responsible ownership models that could be applied to our project and what benefits each could have. Also, we thought more about our final product, do we want to create GM seeds in the end with specific crops, or just have a ‘nitrogen-fixing traits’ that could be used as a technology by others. You can see more on the ownership page and Entrepreneurship page about how we imagine our final idea.</p>
<divclass="h3">National Institute for Public Health and the Environment (RIVM)</div>
<p>During our interview with the RIVM GMO office, we learned about environmental risk assessment and what are the steps for commercializing a GM crop in the EU and the Netherlands. Our main question was what the relevant aspects in the assessment of field trials are and how we can mitigate potential risks connected to our GM plant. We learned that risks and containment measures depend on the characteristics of the GMO and the environment it is grown in and are therefore case specific. So, choosing a plant is essential for specific details. A bioinformatics blasting module was discussed to assess safety better.
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<p>We followed up our RIVM discussion about environmental safety by reaching out to Max van Hooren to get more specific information on safety related to our design. He is a member of the scientific secretariat of The Netherlands Commission on Genetic Modification (COGEM). COGEM is an advisory board that provides advice on work involving genetically modified organisms. </p>
<p>We discussed the environmental safety aspects in more detail such as competitive advantage and genes spreading via seeds. Also, important question was, what design would be best; to genetically engineer the host or not or the question of not making the organelle viable on its own.</p>
<p>Amrit Nanda is the Executive Manager of Plants for the Future ETP which is a Non-profit membership-based organization bringing together academia, industry and farming communities to promote the flow of innovation to market in the plant sector. She helped us learn more about GMO legislation in Europe and what possible changes could be proposed to promote the implementation of synthetic biology ideas like ours. We also talked about how important science communication is for the acceptance of GMOs in the public.</p>
