Our key partners were those stakeholders with both high interest and power to shape our project. These included research centers, industry advisors and regulatory and security agencies. Research centers, such as the Faculty of Biology, of the University of Barcelona, gave us access to valuable perspectives and validated the applicability of our tool in disease-relevant applications.

By consulting these institutions, we aligned our work with academic standards and benefitted from collective expertise beyond individual researchers. Industry advisors like Promega provided practical support, such as the technical know-how, reagents and assay systems and helped us bridge the gap between academic theory and practical implementation.

Industry partners contribute a real-world perspective on the usability of our product. With the early involvement of these key actors early, we secured a reliable source of technical knowledge grounded in extensive global lab experience - an essential foundation for credibility and reproducibility.

Moreover, and given the novelty of our project, it was crucial to contact regulatory and safety agencies to ensure our project met biosecurity standards, reassuring both the general public and institutions. By consulting IBBIS and the iGEM Safety & Security Committee, we were confident that our product posed a low intrinsic dual-use risk, while we simultaneously explored new frameworks for risk assessment and mitigation. Instead of safety as an afterthought, we proactively integrated it into the design process, which can be demonstrated in our Dual-use assessment.

We also worked with stakeholders with high interest in our project but lower formal power. Nevertheless, their insights were paramount in shaping the educational impact and the usability of our product. We worked closely with teachers and schools, where we tested our educational toolkit in real classroom environments. This was fundamental to ensure our project could have an impact beyond the lab.

Their feedback revealed the importance of age-specific framing and modulability, while also ensuring our project could function as a coherent pedagogical resource to inspire the scientists of tomorrow. In order to guarantee scientific rigor and credibility, we reached out to researchers from different fields who, in the best of cases, would become potential users of our technology.

Contacting them was particularly important as innovation often benefits from cross-disciplinary perspectives, thus expanding our understanding of potential challenges and applications we had not considered. Most importantly, these discussions ensured that our tool was not developed in isolation, but rather as part of an ongoing dialogue within the synbio community.

A third group, constituted by powerful but not particularly interested stakeholders, played a key role in the feasibility and prospects for our project. While not directly involved, government agencies and bioethics committees ensure scientific innovations remain within ethical and legal boundaries. These agencies act as gatekeepers who safeguard the public interest and ensure that emerging biotechnologies, such as ours, comply legally and institutionally. By keeping them satisfied, we built trust and credibility, reflecting our compromise of integrating proper ethics and governance into every stage of development. For that reason, we contacted Dr. Gemma Marfany, a recognised bioethics expert, to guide us in the process.

The last quadrant includes stakeholders with low power and direct interest, such as the general, non-specialized public and the media. Although they did not directly influence our project, they remain an essential part of building trust, transparency and social acceptance around synthetic biology. Public perception plays a decisive role in how biotechnologies are understood, regulated and adopted, which remarks the importance of engaging with them.

Through different outreach activities, we made our work accessible to non-specialists, demystifying synthetic biology and showing that it can be developed responsibly and ethically. Complex technologies like RNA-based regulation can easily be misunderstood, which is why we accepted the challenge to take our project beyond academia and foster mutual trust between scientists and citizens.

• Aim: Paul Copeland has been a leading figure contributor in uncovering the mechanisms of selenocysteine incorporation at UGA stop codons. He played a key role in the identification of the eukaryotic SECIS-binding protein 2 (SBP2) and in establishing the mechanistic model in which SECIS works. His expertise was crucial in laying the foundations of our project.
• Contribution: He considered the project exciting and feasible, suggesting tools like XFold and Benchling, testing magnesium concentration ranges, and adding selenium supplementation. He also proposed alternatives such as monoluciferase assays, GFP normalization and in vitro transcription with TNT kits. He highlighted the limitations in quantifying SECIS readthrough and advised proof-of-concept experiments in vitro before moving to cell lines. He kindly provided SBP2 for our use.
• Implementation: We tested magnesium and selenium conditions, explored monoluciferase and GFP normalization approaches, and considered in vitro transcription for validation. However, we will assess feasibility with HEK cells as a starting point.

• Aim: Promega is a leading biotechnology company that offers a wide range of life science products. Several of their tools have been essential during the development of our project, including the TnT Coupled Reticulocyte Lysate System, the Dual-Luciferase assay and the plasmid purification kits.
• Contribution: At the Discover-Glo conference by Promega, we connected with stakeholders such as Irena Montiel and Eric Rovira. Irena, a key account manager at Promega and expert in bioluminescence, advised against NanoLuc or the GloMax Galaxy system for RNA-ligand interactions studies, instead recommending the Dual-Luciferase Assay (Renilla/Firefly), as the most suitable approach for our project.
• Implementation: Based on her advice, we implemented the Dual-Luciferase Assay to monitor frameshift efficiency and evaluate the functionality of the SCR element in our design.

• Aim: Andrew Anzalone is a leading figure in modern genome editing, developing a prime editing technology. His prior experience working with theophylline riboswitches, like those used in our project, made him an invaluable candidate source of feedback.
• Contribution: When our experiments with HEK293T cells failed to produce the expected response to theophylline, we hypothesized that the aptamer might not be folding correctly inside mammalian cells. Upon consulting Dr. Anzalone, he confirmed that he had encountered the same issue: in his work, HEK293T cells did not yield positive results, whereas yeast cells did. Together, we concluded that the discrepancy arises from temperature differences. The aptamer folds correctly at around 30 °C (in vitro and in yeast), but at 37 °C (mammalian conditions) the mRNA misfolds, disrupting functionality.
• Implementation: Dr. Anzalone’s feedback was pivotal, prompting us to prioritize in vitro validation of our construct before moving to mammalian cell culture. Once a solid proof of concept is established, we can then explore strategies to overcome the temperature-dependent folding limitation and adapt our riboswitch system for mammalian applications.

• Aim: Dietmar Funck is a researcher in plant biochemistry and physiology at the University of Konstanz, with knowledge in riboswitch-based regulation in plants.
• Contribution: He noted that the most effective strategy is combining two riboswitches responsive to the same ligand within a single transcript. He emphasized the challenges of predicting riboswitch efficiency for specific genes, as affinities highly depend on sequence context, and remarked on the limitations of in silico models due to the complexity of in vivo RNA folding prediction. Overall, he underscored the importance of empirical testing over sole reliance in computational models.
• Implementation: Dietmar’s insights were crucial in helping us recognize the practical limitations of our project and the potential discrepancies between computational predictions and actual lab outcomes. His guidance reinforced the necessity of combining predictive tools with hands-on validation, ensuring a more robust approach to riboswitch-based regulation.

• Aim: Dr. Eric Rovira is a geneticist and RNA engineer, featured as a speaker at the Discover-Glo conference. His research focuses on RNA structure tools to investigate both fundamental and applied aspects of gene regulation, with particular emphasis on human gene expression. His extensive background and riboswitch expertise provided us with valuable insight.
• Contribution: In an interview, Dr. Rovira discussed riboswitches in personalised medicine and gene therapy, stressing design considerations such as linker placement and SECIS modifications. He noted guanidine as a potential ligand, suggested post-SELEX software for candidate identification, and highlighted SHAPE-MaP as a tool for functional screening. He expressed strong interest in the team’s riboswitch work and its future applications.
• Implementation: While not all suggestions fit the project’s scope, his advice was highly valuable. Our PI confirmed that our luciferases were suitable, and we validated that our SECIS element required no modifications. We chose not to pursue guanidine as a ligand, favoring the well-characterized theophylline aptamer, and we set aside SHAPE-MaP due to time constraints. Nevertheless, Dr. Rovira’s input broadened our understanding of riboswitch applications and highlighted promising directions for future toolkit expansion.

• Aim: Dr. Roger Castells-Graells leads the Biomolecular Design and Structural Nanomedicine group at the Spanish National Cancer Research Center. As an expert in protein design and structural biology, he was well-positioned to give valuable feedback on our toolkit and its potential applications.
• Contribution: Thanks to his expertise in bioinformatics, Dr. Castells recommended using Boltz for small-molecule predictions and highlighted the limitations of AlphaFold3 in modeling theophylline interactions. He guided the team in visualizing theophylline–aptamer interactions with ChimeraX, validated the Tadpole software, and emphasized evaluating potential toxicity of theophylline and SECIS elements. He also noted that the toolkit could benefit his own research on protein cages by enabling precise control of protein size to avoid steric clashes and aggregation.
• Implementation: His feedback allowed us to improve and adapt the Tadpole software, and set realistic expectations for its predictive capabilities. Although advanced ligand modeling was limited, his guidance on visualization clarified our development roadmap. Addressing his concerns about theophylline toxicity, we performed a HEK293T assay to determine a safe concentration range for our experiments.

• Aim: Atsushi Ogawa is a recognised expert in the field of ligand-responsive RNA switches. His research enabled ligand-induced translation by modulating the internal ribosome entry site (IRES) structure, producing both OFF- and ON-type riboswitches. His papers laid the foundations for the early stages of our project.
• Contribution: During the initial stages of our project, we were concerned that unpaired nucleotides in the aptamer might accidentally base-pair with parts of the IRES in the OFF state, thus interfering with the correct folding. Upon consulting Ogawa, he clarified that even if it was a possibility, ligand binding makes the ON conformation more thermodynamically favorable, overcoming the mispairing.
• Implementation: The IRES-based mechanism Ogawa suggested was not compatible with our project, given that the proximity of the aptamer and the switch would probably lead to misfolding. In order to minimize unwanted hybridization, we decided to use a SCR-based model.

• Aim: As an outstanding researcher in Cambridge University with a background in biochemistry and molecular genetics, he represented the kind of profile we expect to use our technology.
• Contribution: He recommended including illustrations and explanations to clarify what our software does, with special emphasis in the SCR section and the Skippit part. He considers that making visible the calculations done with ViennaRNA is key to ensure trustworthiness and give credit where it is due.
• Implementation: We redesigned the website with illustrations and diagrams to facilitate understanding.

• Aim: Jacob González is a PhD postgraduate student in the Genetic and Epigenetic Inheritance in Plants Group in the University of Cambridge. Specialized in bioinformatics, he could detect our weaknesses and propose strategies to improve our toolkit.
• Contribution: Jacob highlighted the need for a clear user guide and a graphical abstract to illustrate the workflow. He also recommended a user-friendly interface where the inputs are inserted and the web returns the linker. He advised improving default parameters for quick demonstrations, adding result analysis features, and providing a single grouped output file instead of multiple documents.
• Implementation: We added new figures and graphs to visualize the code, also including a user guide and tutorials for easier navigation, and ensured transparency by providing documentation on the web and the source code in the GitLab repository.

• Aim: Manuel Reina is a professor of Cell Biology at the University of Barcelona and director of Celltec-UB, a research group specialized in developing and transferring cell technology to partner enterprises, facilitating the translation of academic research into practical applications. His perspective helped us anticipate how our ideas may be applied in real-world contexts.
• Contribution: He suggested mechanisms through which the riboswitch may be applied, for example, inside a bioreactor to regulate cell concentration and trigger apoptosis when it becomes excessive. He also underscored a critical aspect for future application: the half life of our product, given that this parameter is fundamental in defining long-term strategies.
• Implementation: Guided by his advice, we plan to integrate stability assays into our design pipeline, so that future iterations can meet the requirements of large-scale production and scalable implementation.

• Aim: We presented our project in a poster presentation with fellow colleagues and students. More importantly, we had a valuable exchange with another research group working on readthrough.
• Contribution: Their research focuses on retinitis pigmentosa, a blindness-causing disease resulting from nonsense mutations, and explores readthrough as a therapy to restore function of truncated proteins. Even low levels of drug-induced readthrough (～2%), may partially restore vision. Our system achieves readthrough efficiencies above these thresholds, confirming its potential therapeutic applications.
• Implementation: By understanding the applicability of our project in disease contexts, we can optimize our system for future translational studies. It also remarked on the necessity of a functional SCR tool to advance research. On another note, we also received feedback on presentation clarity and poster design.

• Aim: PhD Antonio del Río is an expert in Developmental Neurobiology, Neural Regeneration and Neurodegeneration at the Institute for Bioengineering of Catalonia, with keen interest in prionic diseases. Given that fine-tuning protein expression is a central aspect of our project, we believe he could provide valuable feedback.
• Contribution: He outlined prospective strategies in which our riboswitch could bypass the stop codon and create prion-like proteins, facilitating the study of these challenging molecules. Based on existing prionic biosensors, he suggested a novel strategy to quantify readthrough by placing GFP downstream of the stop codon, such that fluorescence intensity is proportional to translational readthrough.
• Implementation: Building on his suggestions, we could optimize a riboswitch capable of generating prion-like protein variants, expanding the applicability of our tool to neurodegenerative research but also providing a powerful model for studying protein misfolding and aggregation in a controlled environment.

• Aim: We aimed to understand how our toolkit could best support teachers, identifying their needs and exploring how to address them effectively.
• Contribution: He suggested designing a modular project with related activities that could also stand alone, and advised us to highlight the curricular skills developed in each activity to make the toolkit more appealing to teachers. He also contributed to shaping the ethics activity.
• Implementation: We applied his suggestions by making the toolkit modular and specifying the skills developed in each activity.

• Aim: We conducted a pilot test of the toolkit’s art activity to gain direct, first-hand insight into how our idea could be improved.
• Contribution: Although both the teacher and students enjoyed the activity, they felt the time was too limited, and the students expressed a desire to share their animals with the rest of the class. We suggested using ChatGPT to help with the drawings since not everyone draws equally well, but we ran into issues: some groups didn’t use it at all, while others relied on it too heavily and didn’t engage in the creative thinking process.
• Implementation: To address this, we divided the activity into two sessions and added a dedicated space for students to present and share their creations. We also decided to remove ChatGPT from the activity. Although some drawings might not turn out perfect, it’s better that participants do the thinking themselves.

• Aim: We carried out an adapted pilot test of the toolkit’s bioethics activity to gather direct, first-hand insights on how to improve our approach. At the end of September, they tried out the second activity of technology and gave us valuable input.
• Contribution: From the bioethics pilot test we saw that while students were initially in favor of applying the new treatment in the fictional case, their opinions shifted as more complex and nuanced questions were introduced. Regarding the feedback from the technology activity, The teacher recommended adding the history of AlphaFold for context, improving the introduction’s visual appeal, and addressing speed issues. He also questioned whether AI tools like AlphaFold truly solve the structure prediction problem, or if utility should be distinguished from genuine understanding and justification.
• Implementation: To better support the debate in the bioethics activity, we provided key arguments for the positions students had to defend, particularly for the teams opposing the treatment. As for the technology activity, we will work on their suggestions, trying to implement this new philosophical point of view.

• Aim: In order to test the toolkit’s technology and communication activity, we carried out a pilot test to obtain preliminary feedback and identify areas for improvement before broader implementation.
• Contribution: Daina Isard school really liked our toolkit when it was presented to them. They said they wanted to test all the activities and use the toolkit as it was intended, for the moment they have tried the technology and the communication activity, but they will also do the experimental one. The technology activity was deemed a success, greatly enjoyed by the students and allowing them to understand how to use BLAST through hands-on learning. The communication activity, although positively received, was considered somewhat repetitive.
• Implementation: The feedback not only allowed us to refine the activities by reducing redundancies, but also steered us to imagine how the toolkit can be better tailored to different learning contexts. These adjustments ensured greater effectiveness and potential of the toolkit in classroom settings.

• Aim: La Llar de la Recerca is a youth association dedicated to spreading science and technology amongst young people in a catalan region called Penedès, we attended an oratory workshop organised by them and led by Eulàlia Soler, professor of oratory at Universitat Pompeu Fabra.
• Contribution: We drew inspiration from this association to shape our vision of the 360º scientist: someone with a strong cultural background and an awareness of the world around them. In the workshop, Eulàlia explained how debates are structured in official competitions, emphasized the importance of thorough preparation, and shared practical tools to improve debating skills.
• Implementation: We thought of the education plan around this idea, and we adopted a structured debate format for the bioethics activity (introduction, discussion, and conclusion, with questions limited to the discussion phase) and equipped students with some of the tools and strategies introduced in the workshop.

• Aim: Dr. Gemma Marfany is a molecular geneticist and Professor of Genetics at the University of Barcelona. She is also a member of the University of Barcelona’s Bioethics Committee and is widely recognised for her contributions to the field of bioethics.
• Contribution: As a professor and science communicator, Dr. Marfany shared her extensive experience in student engagement and pedagogy. She suggested strategies to improve engagement, underscoring retroactivity between teachers and the team to continuously refine the toolkit. She highlighted the strengths of our activities while also identifying areas for improvement, such as the lack of contextualization between our specific project and the activities.
• Implementation: A key aspect was to define the demographic target of our toolkit. Given that a relative maturity is required to debate the activities, especially the debate, we ponder that ages ranging 15-17 might provide best results. With her guidance, we designed a biotechics activity that allowed even non-biology students to reflect critically on present and future challenges. Related to the DNA extraction experiment, Gemma suggested changing the media we use to spark scientific curiosity. This adjustment, along with her pedagogical insights, will allow us to strengthen the overall educational impact of the toolkit.

• Aim: The Cosmoxarxa is a volunteer network at CosmoCaixa, Barcelona’s science museum, that meets regularly for conferences and discussions. It offers a space where ideas can be discussed with people from diverse backgrounds.
• Contribution: We presented our project and explained our aptamer-riboswitch model. Participants raised questions raising topics such as safety and potential therapeutic applications, while medical students in the audience expressed interest in its prospects for gene therapy.
• Implementation: The presentation highlighted which aspects of our project sparked more interest and which ones were harder to explain, helping us refine our communication of synthetic biology. This insight improved the accessibility and clarity of our explanations for the general public.

• Aim: Ina Kruger and Kathryn Dungey are postgraduate students in the Genetic and Epigenetic Inheritance in Plants group at Cambridge University. They have conducted activities to spread science, from which we could take inspiration from.
• Contribution: Our education toolkit includes a hands-on activity of DNA extraction from fruit and visualization, aiming to familiarize the general public with the basics of genetics and biochemistry.
• Implementation: In response to our inquiry for advice, they recommended adapting the protocol by preparing intermediate products and pacing the activity for smoother execution during discussions. They also suggested improving accessibility by linking concepts to participant’s own experiences and explaining the scientific role of each reagent, so that the activity may be replicated at home.

• Aim: The International Biosecurity and Biosafety Initiative for Science is an organization devoted to act as a consultor and assessor in synbio projects. We contacted Technical Consultant Lucas Boldrini, given that his prior experience with iGEM familiarized him with the specific requirements of such projects.
• Contribution: They reassured that we may be overthinking, which is ultimately positive for ensuring proper use. They considered the current dual-use risk is low, emphasizing that the sequence we provide is innocuous and danger lies in the payload expressed with it, but that’s beyond our scope. As safeguards, they recommended focusing on educating users, restricting applications to non-pathogenic chassis, and considering consumer screening as a preventive measure.
• Implementation: Following the IBBIS philosophy and its open-source Common Mechanism screening tool, we adopted a similar open-access approach, valuing its benefits over potential risks, and we developed a client screening method. On their advice, we also sought further evaluation from the iGEM Safety & Security Committee for further analysis.

• Aim: We consulted the iGEM Safety & Security Committee about potential dual-use concerns related to publishing Tadpole software as open source. To ensure responsible action, we submitted our Dual-Use Assessment based on frameworks from the Netherlands Biosecurity Office and the US National Academies of Sciences, Engineering, and Medicine (NASEM).
• Contribution: The Committee congratulated us on our careful assessment and concluded that our technology does not pose an intrinsic dual-use risk. The primary concern lies not in the regulatory logic itself, but in the downstream payload that could be expressed if safeguards were bypassed. However, the Committee reassured us that current biosecurity practices are sufficiently robust to detect such risks, and that effective medical countermeasures are available.
• Implementation: Now that we were confident that our software and sequences could be safely shared and published, we decided to apply for Best Composite Part. Their feedback also empowered us to engage in discussions on risk assessment and to better anticipate how future technologies might be misused.