In the Human Practices (HP) part of the project, our team aimed to assess the impact of our project on the world. We considered the environmental, social and economic impact. Since our idea is bold and revolutionary and uses synthetic biology in agriculture, it was important to us to shape our design in a responsible way and think of all the potential risks in case our technology was to be implemented in society. Hence, we applied the notions of Value Sensitive Design through stakeholder interviews.

We started by using the Value-Sensitive Design (VSD) 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. [1] 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.

The steps of our HP and Integrated HP approach, based on the VSD analysis, are shown in Figure 1. The VSD consists of three main phases namely the conceptual, empirical and technical part. We applied these stages in our HP work. In the conceptual part, 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 empirical part 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 technical part 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.

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.

We are a team from the Netherlands and even though many of us are international students, we care about the environment we live in. In the Netherlands, pollution from reactive nitrogen deposition is a major problem and immediate action is needed in the short and long term to restore nature and allow new economic activities to be pursued.[2] We felt an obligation to find a solution that could help local people and the driving agricultural sector of the Netherlands. [3] A sustainable solution for agriculture is not only important locally but globally as well. There is a growing global food demand by rising populations where agricultural productivity must be doubled by 2050 to feed the world.[4] However, since sustainability in agriculture is already a challenge, the question is how we can achieve a drastic increase in productivity sustainably?

We chose a synthetic biology approach to answer this question. We were looking for a solution that is environmentally and socially sustainable, that helps solve food security problems, is accessible and of course safe. This is a big task and at the beginning of our project we were wondering what if our idea is merely a techno-fix? This means that while a technology serves as a solution, it mostly addresses the (unwanted) effects, rather than the root of the problem. [5] This is the question where our Human Practices (HP) and Integrated Human Practices (IHP) journey started.

Done Scott describes philosophical and practical criticism of technological fixes in his article “The Technological Fix Criticisms and the Agricultural Biotechnology Debate”. [5] He summarizes that “The practical criticisms of technological fixes serve as a warning against the inherent dangers of addressing complex, multifaceted problems with narrowly conceived technological fixes. The philosophical criticisms seek to undermine a worldview that sees technological fixes as the primary means to advance civilization and social welfare”. He lists three often brought up arguments against techno-fixes that are 1) they don’t serve as a true solution 2) they only create more problems and 3) technological fixes preserve, or fix, systems that should be abandoned in favor of better alternatives.

We are aware that our solution might not solve the social and political challenges underlying nitrogen pollution and food security. But it can clearly serve as an extra option for different actors to use to tackle above mentioned challenges. This gives more time to deal with the root problem. As Dr. Britte Bouchaut – who is an Assistant Professor at the Safety & Security Science group at TU Delft – mentioned in her presentation at the Dutch iGEM meet 2024 (organized by The Centre for Living Technologies and supported by iGEM WUR and iGEM TU/e) it can be okay to design a techno-fix, as long as we are aware of such, and think about the impact and the consequences of our technology. This way we can respond to the three practical critiques listed.

Putting this into practice, we assessed the environmental and social impact of our synthetic biology idea, we talked with relevant stakeholders and implemented the information we learned in our project by making design choices based on the input we got and on our VSD analysis. (see IHP page).

Therefore, we believe that, although our idea can be regarded as a technological fix, it can serve as a great (temporal) solution that has been designed responsibly.

As mentioned in the Our Responsible Innovation section, we used the VSD (Value Sensitive Design) analysis as a tool to guide our design process, ensuring it is both responsible and centered on human values. This approach translates values into technological norms and design requirements. By creating value hierarchies, we make the decision-making process behind our design specifications more transparent, especially to external stakeholders. A values hierarchy (see Figure 2) consists of values—principles that promote the common good, such as freedom and sustainability—and norms, which are the rules for achieving those values. The most relevant norms are end-norms, which can also be viewed as objectives, goals, or constraints.

In the conceptual phase (see Our responsible innovation section) of our VSD we thought how our nitrogen-fixing plant (or self-fertilizing plant) plant would contribute to the problem.

The engineered plant would require little to no nitrogen fertilizer, which would prevent soil acidification and reduce ammonia production, thereby lowering CO₂ emissions. Additionally, there would be minimal or no reactive nitrate leakage into freshwater bodies and coastal regions, helping to protect the environment and biodiversity. Fewer nitrogen oxides would be emitted into the atmosphere, contributing to a reduction in greenhouse gas emissions.

The reduced need for fertilizer would lower growing costs globally, especially given the dramatic rise in fertilizer prices in recent years. [7] This impact would be even more significant in countries with lower food security and limited access to mineral nitrogen fertilizers. At the same time, theoretically, crop yields would remain high compared to conventional fertilizer use, allowing for more sustainable food production to meet the demands of a growing population.

Now we can briefly return to our question ‘Is our idea a techno-fix?’ and how we can respond to the practical criticism on techno-fixes. With the benefits listed above we thought a potential nitrogen-fixing crop could serve as a solution to the narrowed nitrogen pollution and sustainable agriculture problem described before. This answers to the first practical criticism listed in Scott D’s paper that says techno-fixes don’t serve as a solution.

Stakeholders are all individuals or institutions that have an interest connected to our self-fertilizing plant technology. Below is a power-interest grid with the most important identified stakeholders associated with our project in the Netherlands. Stakeholders were identified based on literature review. Their position on the map represents how much they are affected by the nitrogen problem and our solution (interest) and how much change they can achieve to solve the problem (power).

The identified/relevant values were food security, accessibility, social/environmental sustainability and safety. The values hierarchy of the two most important values: safety and accessibility can be seen in Figure 4 and Figure 5 as an example.

Safety was found to be an important value for the European Union but also for 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 to satisfy those norms in our design, such as ‘the genetically modified (GM) plant shouldn’t outcompete native species’. 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.

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.

See what design adjustments we made regarding these questions after interviews.

When our team came up with our initial approach and design, it was very exciting and seemed like a great solution that the Netherlands and the world could hugely benefit from. Talking with Tyler Coal and Jonathan P. Zehr helped us with our first design idea. The first big question was on the feasibility of the project. Thus, we developed a roadmap outlining the essential steps required for a crop plant to successfully incorporate the nitroplast organelle and fix nitrogen from the air. To discuss our approach and receive a critical view we talked with scientists from seed companies like KWS Seeds. Then our concern became that we are creating a GM plant by means of synthetic biology, and whether that would really be a great solution or a techno fix? (see the team's standpoint on this at Our responsible innovation approach) To answer this question, first, we talked to Martijn Schaap from TNO to learn more about the nitrogen problem/pollution in the Netherlands. Then we contacted RIVM and Max van Hooren from COGEM to talk about environmental safety and what measures could be applied to our project. We also discussed aspects of risk assessment. Then we had a discussion with Amrit Nanda, who is the Executive Manager of Plants for the Future ETP on how our idea could be applied in Europe and how to communicate our project since GMOs are not popular in Europe currently. Meanwhile we talked with dr. Zoë Robaey (WUR) about responsible innovation and the social impact of our project.

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.

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.

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.

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 section.

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.

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.

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.

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.

As a follow-up to our RIVM discussion about environmental safety, we were redirected 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 independent advisory board that provides advice on work involving genetically modified organisms.

We discussed the environmental safety aspects in more detail such as competitive advantage and genes spreading via seeds. Also, another 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.

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.

This helped us improve how we present our project to the public during different public activities. We talked about the difference in GMO legislation approach between Europe and other countries. Resulting, we discuss a potential approach how in Europe the application of GM crops could be looked at.

Disclaimer! Statements made during this interview are the personal opinions of Amrit Nanda and do not represent the positions of Plants for the Future or its members.

Reaching out and talking to stakeholders was always in a respectful manner. The team applied for the approval of our project by the TU Delft’s Human Research Ethics Committee (HREC) since our work involved Human Research Subjects. For this we needed to fill out a form and identify potential risks connected to our activities involving Human Subjects (interviewees), create a data management plan approved by the Faculty Data Steward and write an Informed Consent Form. Prior to all interviews, the Informed Consent Form was sent asking for consent for posting and eventually sending the interviewees the transcript of our conversation for their approval for sharing the information on the wiki webpage.

In our IHP part we tell how we integrated the ideas from the interactions with stakeholders. You can read about each interview and what we learned from it in Stakeholders we talked to.

We prioritised the value environmental safety in our product design. First it must be noted that a GM nitrogen-fixing crop engineered with our idea wouldn’t be possible to cultivate in the Netherlands or Europe according to the current legislation. This we discuss in more detail at the legislation part. Even for field trial experiments it could be hard to get a permit. Regardless we wanted to know more what environmental safety measures we would need to think of or implement in case we would have our technology ready for testing. We would be obliged to conduct an environmental risk assessment if we aim for commercialisation of our product on the market.

What we learned from the interviews with RIVM and Max van Hooren from COGEM that environmental safety – including containment measures for field trials – of GM crops or plants is very case dependant. For instance, it is important to know what plant in which environmental conditions we would want to grow. This is relevant for two main reasons; one is the crossing of our GM crop with native species therefore spreading the genetic information, the other is the spreading of our GM crop in the environment. In the light of these two aspects, we had to think of the target country and location. Originally, we were thinking of the Netherlands where there are relatively harsher winters so for example corn couldn’t possibly survive them and spreading of it wouldn’t be a problem. Neither cross pollination because there aren’t many relative species native to the Netherlands. However, rapeseed would, for example, be a poor choice because it is widely cultivated in the Netherlands and there are native species as well.

Additionally for a market application we need to characterize all genes and inserts in our final GM plant and examine the genetic information surrounding the inserted genes. For this we should look at bioinformatics (like blasting ) for toxicity and also compare if the plant would perform or be similar to the wild type. At the same time, we should look for possible mutations in the gene and around the gene so that we know if any unwanted characteristics appear

When talking about the spreading of our GM plant in the native environment, the main reason could be the competitive advantage of our plant compared to the wild type species , as Max van Hooren highlighted. There is good reason that a plant that can efficiently assimilate nitrogen from the air and is independent from the nitrogen assimilation in the soil by bacteria can outcompete those that don’t have this trait. We thought this could be especially relevant in regions where the soil has poor nitrogen content. This carries a risk that our GM plant becomes invasive and can cause the extinction of native species thus would disrupt the balance of the ecosystem. However, if we work with crops, this aspect could be less relevant because crops usually require extra nutrients, pesticides, and herbicides compared to wild relatives.

Another important aspect of spreading of our GM crop plant in the native environment is the potential spread of its genetic information. This is important because wild type species could acquire the foreign genes.Counterargument could be that this could happen naturally as well but we are directly introducing a trait that normally could require millions of years of evolution. This could happen by gene transfer or cross breading by pollination. Gene transfer between plants happens very scarcely.[9] We therefore focused on cross pollination between the wild-type relatives and the GM crop. We discussed with Max van Hooren that if the nitroplast would be successfully incorporated as an organelle, probably the pollen would not contain it so the nitroplast itself wouldn’t spread by pollination thus nor the nitrogen-fixing trait. However, if the host organism itself also has inserted genes to have the ability to incorporate the nitroplast, then these inserted genes would still be transferred by the pollen to native relatives.

So, based on all derived information, we concluded that for containment measures regarding the design of our GM plant, it best would be that 1) the host doesn’t have foreign genes inserted that could spread with the pollen . Then the organelle being transferred by pollination introduced wouldn’t be a concern 2) the organelle wouldn’t survive on its own . This could mean that it is dependent on the host so it cannot be transferred easily. This is inherent to our original design. Additionally, we could test the spreading of genes by cross pollination with direct crossing experiments between the wild type plant and our GM crop .

Another safety measurement option is either a genetic modification added so that the seeds are not viable or make a hybrid seed , so the resulting plant is infertile. These methods haves been applied before by companies like Monsanto and raised many ethical questions although mostly due to their commercial model. [10]

When we were thinking about uncertainties related to GMOs, we were told by RIVM that the EU Directive 2001/18/EC [22] on deliberate release of GMOs requires that risks are reassessed every 10 years after a product is on the market. This means monitoring of the cultivated GM crop to check unforeseeable affects. In order to have more meaningful data and to be able to track the spread of the genetic material of our GM crop we thought to engineer a marker into our host plant and our nitroplast. This could be important also for traceability of our GM crop in the food chain. [11]

‘Thinking of ownership, patenting and who will use and how our GM seed product is part of responsible innovation. ’

We were curious how we can manage the questions above responsibly. dr. Zoë Robaey explained that the question of patenting always comes to mind when we think about GM seeds. We wanted to design our project in such a way that it is accessible so the ones in need can benefit from it like farmers that don’t have access to mineral fertilizer [12] or for farmers or governments that could use it in regions where reactive nitrogen emissions are a big threat for the environment.

However, the first question is who is going to bear the costs of the development of nitroplast seeds and what will the developers ask in return? We read about cases where the seed sector and GM seeds caused legal and ethical issues. [8], [13] Zoë emphasised the problem by raising the question if only rich farmers can pay for our technology, ‘we just create a new socio-economic problem.’ We know that the development of such nitrogen fixing plants is costly and would take at least tens of years of research [14]. So how do we make this seed available at a reasonable price that farmers could benefit? Zoë Robaey suggested that ‘We should make our design in a way that it gives something to both farmers (the public) and the ones that create it’. This could be achieved by implementing an appropriate ownership model or by developing partnerships and cooperating with public actors . However, we must select the plant we want to engineer and look who are in charge of that crop.

Thinking about ownership is important not just because of the value of accessibility but also when it comes to thinking about responsibility. Zoë Robaey in her review paper ‘Rethinking ownership of genetically modified seeds’ describes that preventing costs and consequences from the uncertain risks of GM seeds can be achieved by a different set-up in the distribution of rights and responsibilities then current usual ones where solely a company owns the seed with a patent on it. Difference can be achieved by involving users like farmers and not just scientists or the company that created the GM seed as owners and by stating that all users have forward looking moral responsibility to reduce environmental and societal risks from the use of the GM seed. Key point here is that owners have access to knowledge about the seed and to create cooperation among different owners. Zoë mentioned that the case of the introduction of GM eggplant in Bangladesh is a good example for a alternative ownership model where GM seeds are indeed owned by multiple actors at the same time as the pest resistant technology was donated to a public research institute, and the seeds were given to farmers. Additionally, farmers can keep and re-use seeds, may be even continue breeding them. [15], [16]

For implementing an adequate ownership model for our project, is a similar one that happened in Bangladesh. Another possibility could be that we patent our technology so there is return for our investment, but we also provide free licenses to potential partners which could be NGOs or local research or breeder institutes. A good example for this model is how Wageningen University handled there CRISPR Cas technology. [17]

Additionally, a different approach that Zoë brought up is to make our project open source. This is a relevant idea because of the scope of this project thus collaborations between different research groups is needed. This way there would be free access to information on how to develop such nitrogen-fixing plant trait and the trait could be faster adapted by researchers to different local weather conditions in different crop varieties and the needs of farmers. This way the return on investment could be managed by donations or partnerships. One example of such an open source is the iGEM wiki. An idea is that we can use the iGEM wiki as an open-source platform to help people learn from our project. There has been other attempt for adopting the open-source model for seeds such as the Open Source Seed Initiative (OSSI), which is an organisation dedicated to maintaining fair and open access to plant genetic resources. An option is to adopt the license of the Open Source Initiative for our project.

We also learned from talking with Zoë Robaey that for the social impact of our project traceability of the nitroplast GM plant is very important. Traceability is relevant for food safety and environmental safety. To detect our GM plant easily one approach could be to engineer the expression of a marker gene that would be more abundant so it’s detection would be easier.

When talking with Amrit Nanda, she advised us that if we want to implement our idea we should focus outside of Europe. There have been very strict rules on genetically modified organisms (GMOs) since the 2000s and after the adoption of the GMO Directive, no GM plant variety has been approved for cultivation in the EU. On the other hand, we were curious what new regulations could be proposed to change that situation to bring our idea to Europe? Amrit explained that in the EU, the approach to starts from the perspective that a GMO has potential risks on the environment and health and the benefits are not looked at. It is very expensive and cumbersome to conduct enough studies to prove that the new GMO does not pose a risk. Therefore, small companies cannot afford to create GM plants for example that could be useful for society. It is important to mention that we are talking about cultivation here, not import. The EU imports a lot of GM plant products. However for cultivation, there is only one GM plant variety grown in EU and only in Spain and Portugal.

What could be improved is that the focus moves from the technology used, to looking at the final product: the plant variety. The benefits of the variety should be given more emphasis. Good examples of enabling legislation can be found in many non-EU countries, including the USA and Canada. In this way it could be possible for smaller companies to also develop GMOs for the EU market.

We asked the question How to possibly bring about change ? After the conversation with Amrit, we concluded that people should rely more on science and understand the benefits so there would be less opposition to change the strict GMO rules in the EU and its member states. This means there should be more science-communication between research and society. When we asked how we should communicate our projects she said ‘the benefits should be mentioned first. We need to say what can our project help achieve and why does it matter, and why it wouldn’t work with what we already have. People are open to genetic modification if it’s for a greater good.’

We also asked how farmers approach the GM crops in Europe. Farmers are generally not against the notion of GMOs (the ethical side). The possible opposition is more about the ‘practical side than the concept’. The main interest for farmers is the benefits of the plant variety e.g., more resistance to disease or drought. It would be of interest for farmers if we prove that we can reduce the price of cultivation by reducing the fertilizer needed, but only if the price of the seeds was still affordable compared to yield. However, GM plant products need to be processed and separated from non-GMOs because of the traceability and labelling requirements in the EU. This adds extra costs and makes it difficult to sell, as processors and retailers do not want to use or sell such products.

From the discussions and elaborations on different values and impacts we came to some points and designs that we can implement in our idea. Instead of having a final product in our project we are elaborating on a roadmap with the essential steps to create our forseseen nitrogen-fixing crop. Our experiments are providedinformation on the initial two steps of this roadmap that can serve as a foundation for future research lines. Therefore most of the applicable ideas in our IHP work are relevant for a potential product, which we thought to be GM seeds or a potential plant genetic trait. These ideas tell us about how we imagine to mitigate risks that could be social or legal, or environmental problems when this future product is developed. We believe that our idea could serve as a solution for sustainable farming regarding the nitrogen problem . However, we are aware it could be very hard to get a viable crop plant that successfully incorporates the nitroplast and needs less fertilizer than the current breeds while maintaining sufficient yields. Therefore, based on the conversation with KWS Seeds, we thought of an alternative approach that could potentially more easily lead to the same result. Additionally further approaches are also discussed.

Note: in this section some part the term nitroplast is used in general for a nitrogen-fixing organelle, but in other parts of our wiki it solely refers to UCYN-A

To reduce the environmental impact of such nitrogen-fixing crops we thought of the following implementations to be made in our design.

Since Candidatus Atelocyanobacterium thalassa (UCYN-A) the nitrogen fixing organelle also referred to as ‘nitroplast’ and B.bigelowii are not well studied organisms, not much is known about their genome and expressed proteins. However, for a market application all genes and inserts need to be characterized and also after successfully transforming the host organism examining the genetic information surrounding the inserted genes are also necessary. Using bioinformatics tools, we would analyze potential toxicity and possible mutations in the inserted genes and surrounding regions.This could be relevant for environmental and food safety as well. Additionally, this could also help assess how the plant would perform or if it would be similar to the wild type.

To make sure that the GM crop cannot spread easily in the environment we imagine doing crossing tests with closer or further wild type relatives to our plant. This is important because the spreading of inserted genes is unwanted due to the unpredictable the effects on the ecosystem are.

Since no engineering method for the containment of more complex GM organisms exist – such as kill-switches or containment in a gel for microorganisms – a possible solution for the spreading of our plant is non-viable seeds. However, this could raise ethical problems since farmers would have to buy seeds every year, making them vulnerable to the seed provider company as this could lead to exploitation. Also traditional farming practices are violated because farmers are not able to save and improve their seed from year to year. On the other hand, many farmers are already using hybrid seeds that are not GMOs, but do need to be rebought every year to produce high yields.

A big question related to safety that came up during our interview with Max van Hooren is whether the nitroplast should be able to survive on its own outside the host or not? This is a relevant question because if the nitroplast could survive on its own, that means it doesn’t require proteins and enzymes imported from the host plant cell. This implies that there might be a way to only change the plant cell genetic material and not introduce foreign genes. Therefore, the transgenes (genes from non-relative species) will only be in the nitroplast in a contained organelle. The most important consequence of this would be that no foreign genetic material will be spread by pollen hence the nitroplast assembly would only be inherited by the germplasm similar to mitochondria or chloroplast. This would not only make the environmental risk assessment less complicated (and cheaper) but would also significantly reduce the possible risks of cross pollination between native relatives. It must be mentioned that it might occur that genes are adopted by the host cell from the nitroplast as observed before in plastids. [18] This design would go along with the suggestion of KWS Seed scientist to choose a less dependent cyanobacteria candidate for a potential nitroplast so the cumbersome and difficult introduction and expression of the many essential proteins that UCYN-A must import is not necessary.

The other side of the story is that if the nitroplast is independent from the host, there is a chance that it can survive outside the plant, for example, in the soil and potentially spread in the environment. This could have many consequences on, for instance,the alteration of soil microbial communities that also affects the wellbeing of plants. One solution to the spreading of a less host-dependant nitroplast is engineering a kill switch for when it gets out of the host cell somehow due to the change of the environment or the lack of nutrient, the nitroplast starts a cell death mechanism. From the engineering part (incorporation of the nitroplast by fusion) the size problem must be considered as well since other cyanobacteria species are in general bigger than UCYN-A.

We thought of introducing a marker into our GM seed that the plant contains when growing, so we can better track the spread of the genetic material in the environment and the GM seed in the food chain. The marker can be introduced either in the host cell or to the nitroplast organelle itself. The uTP sequence we came up with and tested its expression during our project, could be a marker to look for that indicates the spread of the transgenes introduced into the crop.

Now we can return to our question ‘Is our idea a techno-fix?’ and how we can respond to the second practical criticism which states that techno-fixes only create more problems. By the critical thinking about environmental consequences and by implementing the said ideas could greatly help prevent or at least see clearer the future uncertain environmental effects of our GM crop.

Learning from our HP work, our final idea on how we would handle ownership is to make sure that our developed seed with our proposed technology is owned by multiple actors and all owners act responsibly when using the seed. For instance, during the development by scientists, distribution by companies or organizations, or planting by farmers. This requires that knowledge is available (technical and about safety as well) to all actors and they collaborate and share the information they learn upon the usage of the seed.

This could be done if the development of the GM seed and information on the technology is not owned by a commercial private company but by a local research Institute or university. Examples we could learn from are the cases of the pest resistant Bt eggplant in Bangladesh mentioned before, [16]or the rainbow papaya case in Hawaii where the local university developed a Papaya ringspot virus resistant strain for local farmers . [19]

So to reply to the third practical criticism that techno-fixes are conservative, by rethinking the ownership model, we are truly stepping out of the current industrial agriculture system and hope to implement a more inclusive and accessible technology.

We acknowledge that this could be quite the challenge if the development and risk assessments are too costly to develop a nitrogen-fixing crop with our method since it requires the insertion of multiple genes and an organelle. In line with the entrepreneurship part of our project, we thought of a compromise. If we have the final product as a seed incorporating the nitroplast, a patent on our developed seed or on our method (information) would be filed for return on investment. To make the technology more accessible we would provide the seeds or share the information on engineering with relevant non-profit partners like local institutes or NGOs in Sub-Saharan African regions or in South America or Asia where our technology good help the most.

It is relevant what target crop we choose because it limits where it can be grown and defines the environment that is relevant to ensure safety. This is also important because the EU doesn’t allow the cultivation of GM crops . Therefore, Amrit Nanda advised us that we should focus outside of the EU like USA or Brazil if we want to implement our idea. Potential crops are ones with high nitrogen need and that are mass-produced in the world like maize or sugar beet. If these species are native outside of Europe than it is something to think about from a safety perspective. On the other hand, a design requirement we thought of is that our technology can be easily applied to different plant varieties so local varieties could also have the nitrogen-fixing trait. Similarly, as Elhai J mentions the need of a technology to transplant nitroplasts not only available for the big corporations in his article. He suggests two methods that might work: the regeneration of mature plants after protoplast fusion or transferring the organelle by grafting. Nicotiana benthamiana could be used for this idea, which is known to be a super grafter, able to form grafts with 87% of a wide variety of plant species. [20]

As a first step in the project when working with plants, the approach could be to do experiments first on plant cell cultures or try to modify the germplasm. Max van Hooren advised to choose a model plant organism first like tobacco plant with shorter reproduction time. Or maybe it would be easier to start with a plant that has already symbiosis with n-fixing bacteria so it would incorporate easier the nitroplast like soybean. It could be also a good test for our trait to see if a nitrogen fixing soybean with our approach would have higher yield than a one inoculated with symbiotic nitrogen-fixing bacteria.

We are aware that the nitrogen pollution problem can be approached in many ways from a synthetic biology perspective which made us think critically aboutour project. During our discussion with KWS seeds, the concern was raised that it could be too energy costly for the plant in many situations to have the organelle incorporated into its cell and to maintain it with proteins and nutrients constantly. For example, if there is abundant nitrogen in the soil, then it might not be beneficial for the crop and yields would get lower. Considering this the researchers at KWS suggested that an alternative approach is to not use UCYN-A as an organelle but rather focus on a less host dependent cell or endosymbiont as nitroplast that could provide usable nitrogen for the plant cell. Elhai J in his elaborate article on the potential use of nitroplasts concluded the same.[20] This way, only metabolites would be exchanged between host and organelle and less genes would need to be inserted into the host cell making the engineering process easier.

One possibility is to use cyanobacteria or rhizobacteria that already live in close symbiotic relationship with many plants as nitroplast candidates. This way we think that creating artificial endosymbiosis based on auxotrophies might be an easier task then with UCYN-A. The work by Cournoyer, J.E., Altman, S.D., Gao, Yl. et al [21]creating yeast cyanobacteria chimeras could serve as an example. Their method could be applied to nitrogen fixation instead of CO₂ fixation, eventually being adapted to plant cells and using appropriate bacterial strains.

Another candidate for a nitroplast could be the endosymbiont of the Rhopalodia gibberula microalgae called RgSB that has a genome small for a Cyanothece (close cyanobacteria relative) but 10- to 20-times larger than a chloroplast genome. [20] We contacted Solène Moulin from Stanford University to discuss this idea. RgSB is only lacking the genes for photosynthesis, it has purely metabolic dependency on the host algae so there is no protein dependency. A rough approach could be to try to incorporate encapsulated RgSB’s into a eukaryotic cell (not C. reinhardtii because the size difference is too small) and select cells incorporating the encapsulated bacteria by reducing the ammonia supply in the medium.

Additional approaches that the researchers of KWS suggested are stopping at the level of transforming and incorporating nitroplast into an algae cell that can be used industrially as protein source for example or as organic fertilizer in the field. Then we don’t have to totally reinvent our target crop plant's nutritioning. This is an approach we thought of before, but we think it has less impact. Firstly, because what we learned during other stakeholder interviews is that for industrial algae fermentation, the carbon source in sustainability aspect is more relevant than the nitrogen source. Secondly, this approach would have many safety issues as well such as the escape of the GM algae into the environment and outcompeting native algae species due to its nitrogen-fixing ability. Nonetheless, using our approach in alternative microbial food protein or artificial meat production to cut down on supplying a nitrogen source could be something to consider in the future.

Additionally, what we learned from Martijn Schaap is that in the Netherlands, animal keeping and the manure from the animals are the major root of the nitrogen problem that we are trying to solve. He explained that, for example, the main contributor to ammonia emissions to air is manure. (Emissions into water bodies are a different story). Nitrogen is imported in the form of animal feed like soy and mineral fertilizer. Then the agricultural products like meat and milk or vegetables are exported but the manure is left here containing ammonia leading to an imbalance of nitrogen cycle. He said the aim would be to not import as much nitrogen but recycle it instead. On the other hand, productivity may go down and then food security could be a problem. A synthetic biology solution could be plants that would use the ammonia from manure and converts it into di-nitrogen gas, so creating a denitrifying plan. And we get the nitrogen out of the cycle by using it as isolation material for example.

This section lists future wet-lab experiments that are the next step or additional steps for the project in the future. These ideas either suggested by the researchers at KWS seeds or confirmed our existing ideas.

The idea is to execute further experiments on UCYN-A itself and its viability. The researchers from KWS Seed advised us that doing fusion experiments with a dead organelle is pointless. They suggested to measure if UCYN-A can fixate nitrogen when its alive and outside of the original algae host. Its survival is not possible because UCYN-A lacks essential proteins but it might be a way to go to design a cell-free protein expression system where it does survive (with no guaranteed success). If this experiment has results, then checking the carbon or nitrogen exchange between UCYN-A and the potential host cell could tell a lot of information. A suggestion was to do this by tracking stable 15 N isotope in a cell that contains UCYN-A. Other possibility could be using metabolomics.

As the next experiment to directly test the uTP transit peptide's delivery to the UCYNA-A organelle, the team considered purifying the mNeonGreen protein with the uTP tag from S. cerevisiae. Then injecting it into B. bigelowii by electroporation to observe mNeonGreen localization in the algae cell using fluorescent microscopy.

Next approach would be to perform our fusion experiment with a Chlamydomonas cell that expresses a fluorescent protein (FP), tagged by the uTP transit peptide sequence and track the FP with microscopy. Alternatively, an essential protein for UCYNA-A could be tagged with the transit peptide sequence and fused to a fluorescent protein, allowing for visualization and assessment of the peptide's function.

Techno moral (TM) scenarios are used to describe a possible future scenario in which a specific future application of a novel technology is imagined along with all the consequences, good and bad, it may have. [22] As synthetic biology is a relatively new field, it can be hard to define potential risks and imagine potential future occurrences. Particularly for (young) students, it can be difficult to think of future societal, environmental and ecological effects. In line with iGEM’s mentality, we want to encourage both teams and scholars to think of the potential consequences of their idea.

We were inspired by the work of Zoë Robaey and colleagues on the use of TM scenarios as educational material. They developed a framework for teachers to present and discuss such scenarios that illustrate multiple perspectives on a synthetic biology development, including pros and cons. Considering the novelty of our project, the framework inspired us to develop and write such scenarios, based on our project. By means of such TM scenarios, we want to provide a basis for those who are starting with synthetic biology and help teachers with getting inspired. Therefore, this work is part of the teams’ educational effort in the theme of synthetic biology. The TM scenarios provided below can be used in Step 2 of the provided document by Robaey under the link of the citation. [23]

Scientists created a nitrogen-fixing maize (or other native crop to the region) with the use of synthetic biology. This adapted maize could fixate nitrogen due to having a new organelle in its cells called ‘nitroplast’. The invention was a great scientific achievement! Scientists believed this plant could save millions of people from hunger by providing more food in regions with harsh conditions and lower food security. In addition, growing this maize wouldn't require the use of (expensive) chemical fertilizers, so animals and the environment would benefit from this innovation too – at least, that's what they thought.

The scientists felt great responsibility and thus made sure that their technology was accessible to small farmers as well and not just big companies. They made sure that the technology was not controlled by patents and that it was easily applicable to many other cassava varieties and other crops as well. The scientists made sure that the seeds and knowledge were shared with local governments of countries desperately needing this technology. The seeds were given to farmers and they were allowed to save the seeds and improve their crops. The seeds were indeed performing better, and many farmers that would normally only grow food for their family could now start selling part of their harvest. This also gave more economic stability to multiple regions. Farmers, their families and local governments saw many positive effects of this innovation.

However, after some years, most maize grown in the region contained this nitroplast organelle. This nitrogen-fixing maize caused, although very slowly, drastic changes to the soil microbiome. Due to the plant’s unusual reactive nitrogen usage, and to the organelle’s ability to survive on its own. This first caused native plants to disappear, but soon after multiple species of insects would disappear too and birds started migrating to regions and countries with abundant food (i.e. insects). By the time the relationship between the nitroplast and the effects on the ecosystem became clear, the crop was already so spread that it was impossible to eradicate, or to track the spread of the genetic material. Additionally, some weeds became nitrogen-fixers and started to grow rapidly, again causing problems for farmers and jeopardizing food security...

Scientists created a nitrogen-fixing maize (or native crop to the region) with the use of synthetic biology. This adapted maize could fixate nitrogen due to having a new organelle in its cells called ‘nitroplast’. The invention was a great scientific achievement! Scientists believed this plant could save millions of people from hunger by providing more food in regions with harsh conditions and lower food security. In addition, growing this maize wouldn't require the use of (expensive) chemical fertilizers, so animals and the environment would benefit from this innovation too – at least, that's what they thought.

The scientists were aware that this technology could have many unpredictable negative environmental effects. For instance, they considered that if the non-native genes of this genetically modified (GM) maize would escape, other native maize relatives could inherit the genes and thus alter the ecosystem. Therefore, the scientists designed this new organelle in such a way that it would be dependent on the host maize cell. This means that it would not be able to survive on its own so it wouldn’t be able to spread in the environment. This resulted in the insertion of many genes originated in a marine algae, that originally carried this organelle to provide proteins for. For this reason they made the maize pollen in such a way that if a seed is formed from it, that seed would not be viable preventing the spreading of genes.

The technology got to a seed company that started to produce the seeds. Although the seeds were more expensive compared to the non-GM maize seed varieties, they had same yields after harvest and didn’t need fertilizer. So, in total, a higher income was acquired by farmers. On the other hand, the seeds had to be bought by farmers from the company every year because of the environmental safety measures the scientists thought of (non-viable seeds). This resulted that bigger farms could better afford the seeds and enjoy the advantages more - the rich only got richer. On top of that, these bigger farms were located where there was enough food already. For smaller farmers it was a big investment, but some decided to still purchase the seeds.

However, one season due to climate change suddenly drastic weather conditions happened that ruined the harvest, sending many farmers bankrupt. Still this maize became popular and many countries started to grow it. Then, another season, a new pest turned up and easily spread because all these maize fields were monocultures having the same genetic information. Since the seeds were not viable, it didn’t allow genetic change over generations nor the cross breeding of farmers or breeders. The pest caused a detrimental maize shortage around the world. Then a war happened, and the company started raising the seed prices drastically because of growing energy prices. The farmers who became dependent on the maize variety had no choice than to buy the even more expensive seeds because they already got rid of the equipment for using mineral fertilizer.

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