Is our idea feasible? What is our project good for? Who benefits from our project? What are the ethical and legal constraints of our concept? Every iGEM project needs to consider these questions. To understand how RhyzUp is good for the world and what has to be considered for it to be a responsible project, we reached out to people from different backgrounds from the very beginning. The input we received from stakeholders was key to adapting and reflecting on our project and making it as feasible as possible along with being able to fully understand the potential uses of our engineered Pseudomonas sp. isoF strain.

Since the beginning of our adventure, we knew that we wanted to contribute to the agriculture of the future. We wanted to design a project that would help agriculture become more sustainable while being adaptable to the problems of today and tomorrow. Our special interest in symbiotic relationships between crops and microorganisms led us to stumble across Pseudomonas sp. isoF as a root biofilm-forming bacteria.

At first, we were overwhelmed with questions such as: What strain do we use? How can we improve the symbiotic relationship between our bacteria and the plant? What molecular pathways could we use for our mechanism? How do we test if our bacteria actually benefit the plants?

Dr. Zaira Heredia Ponce is a postdoc in Prof. Leo Eberl's group and our main supervisor in the wet lab. Her research interests lie at the intersection of biofilms, flow-cell chambers, microfluidics, and molecular microbiology. Our initial idea was to overexpress one or two genes that would lead to an increased production of a polysaccharide. However, after our first meeting with her, we learned that biofilm formation could be enhanced more easily by upregulating c-di-GMP levels, a secondary messenger in bacteria. This approach seemed promising, as it allowed us to increase all components and mechanisms involved in biofilm formation. Zaira also helped us decide which bacterial strain to use. We learned that P. sp. isoF is highly competitive and has mechanisms to kill other bacteria strains. With her input, we chose to implement our genetic circuit into P. sp. isoF. She also helped us choose our model plant. Since P. sp. isoF was initially discovered on tomato plants, we decided to work with them to avoid introducing additional unknown variables. Once we began working in the lab, Zaira dedicated time and expertise to supervising and assisting us with all aspects of our experiments, especially the c-di-GMP assay and the CRISPRi system.

Dr. Gabriella Pessi leads a research group in the microbiology department of the University of Zurich. There, she focuses on the molecular biology of Paraburkholderia strains that benefit plants through nitrogen-fixing symbiosis and phytopathogen protection. Dr. Pessi’s expertise in plant-bacterial symbiosis helped us search for the right plant exudate for our sensing system.

Dr. Aurélien Bailly is the leader of one of the sub-teams at Leo Eberl’s research group that focuses on the interaction between bacteria and plants through volatile compounds. In the meeting with Zaira and him, we learned that increasing c-di-GMP levels in the cell is the usual procedure to enhance biofilm production. He recommended insightful papers that helped to determine which of the native DGCs in Pseudomonas sp. isoF were best suited for overexpression and which PDEs were best suited for downregulation.

Under Dr. Joëlle Schläpfer’s leadership, the Schläpfer group is at the cutting edge of understanding the molecular basis of how plants and microbes interact. Dr. Joëlle Schläpfer works with plants and helped us decide which plant experiments were possible in the short time we had.

Vivienne Oggier is a politician and agronomist with a Master's degree in Agricultural Sciences from ETH Zurich. She works at the Agricultural Center of St. Gallen, specializing in vegetable production and beekeeping. We met her at her partner’s farm in the Rhine Valley, where she shared valuable insights with us. We learned about:

• Agricultural challenges and opportunities in the St. Gallen region
• The difficulties posed by increasing weather variability
• Sustainability issues of pesticides
• How Swiss policy on pesticides affects farmers
• The potential benefits of our engineered biofilm for farmers
• The cost of fertilizers
• Potential application methods for our project
• The role of scientific innovations in agriculture
• GMO legislation in agriculture

The discussion with Vivienne Oggier was very valuable for us. Her ideas on application methods for our project were crucial to the development of our Implementation chapter. She suggested several potential applications for our project, including incorporating it as a liquid product during tillage, applying it as microgranules directly to the roots, or adding it as a seed treatment. Direct applications like microgranules or seed treatments would minimize the risk of bacterial spread to the environment.

We also learned that even though the Rhine Valley does not regularly face water shortages, the overall benefits of our project, such as increased pathogen resistance and nutrient uptake, would be of great benefit to farmers. Hearing how the ongoing ban on certain pesticides is threatening Vivienne's partner's yield also showed us the need to develop alternative solutions to pesticides.

Vivienne Oggier was one of the stakeholders who agreed to a recorded interview. You can find the interview in the Human Practices Interviews subpage.

Fritz Meier and his siblings run a modern farming business. The farm consists of three locations with greenhouse farming as well as open-field farming. We visited his farm in Buchs, Zurich, where we received a guided tour and later had the opportunity to discuss with him our questions about agriculture in times of rapid change. We talked about:

• His fossil fuel free greenhouses
• The pros and cons of greenhouse farming versus open field farming
• Current issues facing agriculture
• How water is a major cost in farming. Although he has never had to abandon a field because of water shortages, he knows of others who have.
• How consumers receive information and how they perceive sustainability and genetic engineering
• The importance of microorganisms for healthy soil

Talking to Fritz Meier gave us valuable insights into both greenhouse and open-field farming. During our interview, we realized how important consumers are to the supply chain and ultimately to agricultural practices. We also learned that the use of microorganisms to strengthen crops is possible and important for agriculture and can be implemented in greenhouses and open fields.

Fritz Meier was one of the stakeholders who agreed to a recorded interview. You can find the interview in the Human Practices Interviews subpage

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Andreas Krättli studied agricultural sciences at the ETH and now works at the Gebrüder Meier farm in Zurich. He gave us a thorough tour of the farm and showed us their modern approach to farming. Through his informative tour, we learned a lot, such as:

• How they use microorganisms to improve their crops at the Gebrüder Meier farm
• That it might be easier to use microorganisms in greenhouses than in open fields, where they might run off quickly.
• Modern agricultural practices, such as "hydro salads," which conserve water by using a closed water cycle.

The tour Andreas Krättli gave us was very informative. Much of what we learned was useful for the implementation chapter, where we explore how our P. IsoF strain could actually be used in agriculture. Especially in greenhouses and hydroponic systems, the bacteria could easily be integrated into the closed water cycle.

Dr. Mosisa Wakjira is a Wageningen-based researcher who did his PhD at the ETH Zurich and focuses a lot on rainfall and its implications for farming in Ethiopia. We were able to organize an online interview with him and talked about the following things:

• How water availability impacts Ethiopian farmers in bimodal and unimodal rainfall regions and what the different implications are for these regions
• Water availability issues in rainfed agriculture
• Current and future water conservation efforts
• Global impacts of rainfed and irrigated agriculture
• Global challenges of drought and water usage
• Farming practices that prevent water loss on the field

Our exchange with Dr. Mosisa Wakjira taught us that agricultural practices are heavily influenced by the local climate. He showed us the global importance of rainfed agriculture, which led us to conclude that our root-enhancing bacteria work in more than just irrigated systems and greenhouses. In addition, his comments on current agricultural practices to prevent water loss taught us the importance of integrating our P. IsoF into a system that is well adapted to dry conditions for a holistic approach to drought.

Dr. Mosisa Wakjira was one of the stakeholders who agreed to a recorded interview. You can find the interview in the Human Practices Interviews subpage.

Rahel Emmenegger is an agricultural engineer and the deputy managing director at the Schweizer Getreideproduzentenverband (SGPV). The SGPV represents the interests of grain, oil seed, and protein crop producers. We were able to meet her online for an informative talk, covering the following topics:

• Challenges that Swiss grain producers are currently facing
• How fluctuations in climate influence farmers’ yields
• How Swiss policy on the reduction of fertilizers impacts farmers and the selling prices of their crops
• Which areas in Switzerland are under the biggest risk of water shortage
• What crops tend to be most affected by drought
• Understanding where and when our project could be of use to Swiss farmers
• That the use of microorganisms in agriculture is still a niche market
• That the SGPV sees a lot of potential in the breeding of new, more well-adapted varieties

The interview with Rahel Emmenegger taught us that one of the biggest problems for farmers is the increasing variability of temperature and rainfall from year to year. This taught us that not only too little water but also too much water could be an increasing problem in the future. This highlighted the use of RhyzUp not only for its water retention properties but also for its improved pathogen resistance as many pathogens thrive in wet conditions. We also learned that the broad applicability of root bacteria is important as crops of different genera such as maize, potatoes, and many vegetables are most affected by drought in Switzerland. This supported our decision to use xylose as a sensing molecule since most plant roots exude xylose.

Rahel Emmenegger was one of the stakeholders who agreed to a recorded interview. You can find the interview in the Human Practices Interviews subpage.

Dr. Natacha Bodenhausen is a researcher on soil microbiomes and plant-microbe interactions at the Institute of Organic Agriculture (FiBL). During a Zoom call, we were able to discuss:

• How microbial biodiversity enhances agricultural productivity
• How microbial biodiversity increases stress resistance in crops
• How her research group often uses metagenomics to detect the presence of mycorrhizae
• The importance of ecological testing

Dr. Natacha Bodenhausen advised us to think of a way to test whether our rhizobacteria are present in soils or not. She recommended isolating a genetic sequence specific to our strain to detect it in metagenomic analyses. She also told us that it would be difficult for our bacteria to establish in fields with high biodiversity. On the contrary, she told us that the introduction of new microorganisms could be particularly useful in disturbed or artificial environments with low microbial diversity, such as fields next to chemical plants.

Dr. Anna Deplazes Zemp first specialized in molecular biology and biochemistry, where she obtained her PhD, before taking a second degree in philosophy. She is currently a lecturer and researcher in bioethics and environmental ethics at the University of Zurich and the ETH Zurich. During her career, she has published essays on the ethical considerations of synthetic biology. During our conversation we discussed:

• Biocentric views on the synthesis of life
• The role of naturalness in biocentrism
• The value of bacteria
• Whether, according to some biocentrists, bacteria have interests
• How some ethicists argue that biological functions represent interests
• If the inclusion of a kill switch conflicts with the interests of bacteria
• Ethical considerations of releasing genetically engineered organisms into an ecosystem
• The value of biodiversity and its conservation
• Our responsibility as scientists

The interview with Dr. Anna Deplazes Zemp made us reflect a lot about the ethics of our project. We learned that as long as we are able and willing to explain the introduction of a kill switch, its implementation could be justified from a biocentric point of view. We could argue that increasing biosecurity and preventing ecological spillovers is reason enough. In addition, the benefits to humans and the sustainability efforts of our project must be considered in the overall scheme of things. It also reminded us of the importance of considering ethics, biosafety, and research independence in a project like ours.

Dr. Anna Deplazes Zemp was one of the stakeholders who agreed to a recorded interview. You can find the interview in the Human Practices Interviews subpage.

Claudia Vaderna is the executive director of the Swiss Alliance for a GMO-free agriculture (SAG), an association that raises questions on recent advancements in genetic engineering and wants to promote an open discussion about the applications of genetic engineering. The Alliance is generally critical to the use of GMOs in agriculture and, in collaboration with other associations, has formulated conditions for the regulation and legislation of GMOs in agriculture in the form of a national initiative to ensure the coexistence between GMO and non-GMO crops. Talking to Claudia Vaderna, we were able to understand the counter-arguments to the agricultural applications of genetic engineering, which helped us to reflect on our own project. We learned that:

• Certain applications of genetic engineering (such as herbicide resistance in crops) have been damaging the ecology of agricultural land and natural ecosystems.
• Patent rights in Europe for genetically modified seeds could increase seed prices and harm traditional breeding by through limiting access to genetic resources.
• Coexistence between GMO and non-GMO crops is important if GMOs are legalized for cultivation in Switzerland.
• Products containing GMOs should be labeled as such for full consumer transparency.
• It is crucial to study the impact of genetically modified rhizobacteria on field ecology and biosafety.

The exchange with Claudia Vaderna showed us that we have a responsibility to study the biosafety and the ecological impact of our project. For GMO and non-GMO crops to coexist, our bacterial strain would have to be maintained in the field without the risk of entering neighboring (agro-)ecosystems. It also showed us that a potential product would have to be tested for its impact on soil biodiversity before being applied in the field. We were vindicated in our decision to incorporate a sensing mechanism and kill switch into our bacteria to ensure biosafety, but we learned that we would need to conduct a lot of safety testing before we could responsibly apply a product like RhyzUp.

Claudia Vaderna was one of the stakeholders who agreed to a recorded interview. You can find the interview in the Human Practices Interviews subpage.

Dr. Jürg Niklaus is a lawyer specializing in agricultural law, among other areas. As president of the association "Sorten für morgen" (“Varieties for tomorrow”), he is committed to an open debate on new genetic breeding methods in agriculture and advocates differentiated testing of genetically modified seeds without foreign DNA. His goal is to bring a scientifically differentiated and objective understanding to the legislative process on GMOs in agriculture in order to move away from the so-called moratorium, which strictly prohibits the cultivation of GMOs in Swiss agriculture. We learned from him:

• How GMOs are regulated in Swiss agriculture
• What the core interests of the association “Sorten für morgen” are
• That it makes a difference to the association whether a plant contains transgenic material from other species or not (division into cis- and transgenic)
• That the Federal Council was actually requested to draft a decree for a risk-based authorization regulation for the agricultural plant cultivation of GMOs without transgenic genetic material by mid-2024. This has not yet happened.

The discussion with Dr. Jürg Niklaus was very informative as we learned that for many people there is a difference between transgenic and cisgenic methods and that these methods should be regulated differently. We also learned that currently our project could not be legally implemented in Switzerland due to the moratorium.

Angela Bearth is a social psychologist with a PhD in risk research. Part of her research at ETH has focused on consumer perceptions of genetic engineering and genome editing in food. We met her for a talk in one of the ETH buildings in the center of Zurich. She explained to us:

• How Swiss consumers perceive genetically modified food
• How consumer opinions are formed and how mental shortcuts (i.e., heuristics) play a role
• Exactly what concerns consumers about genetic engineering in crops
• How seeing a relevant individual or societal benefit in genetic engineering increases consumer approval.
• How laws and regulations on GMOs might influence consumer opinions on GMO foods in different countries
• That bacteria are perceived as dangerous by consumers
• That there is currently no freedom of choice for people who want to welcome innovation in agriculture (i.e., new genomic techniques with a specific benefit)

The interview with Dr. Angela Bearth was essential for us to understand what obstacles would have to be overcome for our project not to scare consumers if it were to be implemented. Since our project combines two things that tend to scare many consumers, synthetic biology and bacteria, a lot of education may be needed for our product to be accepted by the general public. This demonstrates the importance of our educational efforts. See theEducation subpage for more information.

Dr. Angela Bearth was one of the stakeholders who agreed to a recorded interview. You can find the interview in the Human Practices Interviews subfolder.

With his experience at Andermatt Biocontrol Suisse, a company that sells solutions containing rhizobacteria for better plant growth and resistance, among other products. Reto Flückiger was able to teach us:

• How products containing rhizobacteria can be applied in the field and in greenhouses.
• The target audience for such products
• That there is also a demand for similar products in horticulture
• That storage is a challenge with Pseudomonas strains
• How the market has developed over the last 20 years

Our exchange with Reto Flückiger was very helpful in understanding the range of application methods of a potential product. We also learned that agriculture is not the only possible application for RhyzUp, as Andermatt Biocontrol Suisse also has rhizobacteria-based products for horticulture. The discussion helped us to understand the demand for products similar to the project we are currently working on. It was particularly helpful in writing the chapter on entrepreneurship and implementation.