Reflecting on Design Decisions
From the very beginning of our project, we committed to examining not only the technical feasibility of our experimental workflow but also the implications of every design choice we made. For example, when purifying DNA, we had the option to use ethanol precipitation, a well-established and cost-effective technique. However, ethanol is both flammable and hazardous when handled in large volumes, and precipitation often yields variable results. After careful discussion, we decided to use column-based purification kits wherever possible. This decision prioritized reproducibility, safety in the lab, and a reduction of chemical hazards, even though it required higher costs.
Similarly, in choosing how to obtain our intrinsic factor (IF) genes, we were confused between assembling them in-house or ordering commercially synthesized, codon-optimized sequences. While in-house cloning would have been less expensive, it would have exposed us to higher risks of error and contamination and would have required significant amounts of reagents and time. By outsourcing gene synthesis, we compromised on flexibility but gained reliability, efficiency, and a safer workflow. These are examples of how our design decisions were not static but iteratively revisited in weekly team meetings. At each step, we consciously weighed whose needs were being served; whether patients, lab members, or the scientific community, and what values (safety, cost, accessibility, reproducibility) were being prioritized.
Responding to Human Practices Work
From the very beginning, we recognised that such a project required careful attention to safety considerations. This was reflected in our Human Practices work: in our interactions with stakeholders and our reflections on the ethical implications of developing the foundation for a new oral supplement.
When conducting our survey, we ensured full compliance with the EU’s General Data Protection Regulation (GDPR). The questionnaire was completely anonymous, and participants were provided with a clear privacy statement detailing how their data would be used and stored. They had to explicitly consent before participating, and all data collected was kept strictly confidential and deleted after use. Questions related to gender, diet, or other potentially sensitive topics were optional, allowing respondents to skip any item they did not feel comfortable answering. Similarly, when conducting interviews with patients, professionals, and stakeholders, we made sure that participants were informed about the purpose of the discussion, that their contributions were voluntary, and that they could withdraw or decline to answer at any time. This ensured a respectful, transparent, and secure research process that protected all participants’ rights and privacy.
Beyond our social research, we also considered safety in the scientific and ethical dimensions of our project. Developing an oral supplement like AbsorBuddy required particular attention to biosafety and human health implications, as it is designed to be ingested. We therefore conducted thorough background research on the safety of our solution and sought extensive feedback from experts, including doctors. Their insights helped us identify potential risks, from immunogenicity concerns to regulatory classification issues, and to design our project.
Approaching Limitations with Integrity
At every stage, we recognized that our project was ambitious and that our team had finite resources, time, and expertise. Rather than overextending, we deliberately mapped out a staged roadmap for functional validation of IF variants. While the gold standard would be a full cellular uptake assay where B12 absorption is linked to cell survival, we acknowledged that such an assay would exceed what was achievable within our iGEM cycle.
Equally important was our effort to situate our work within the existing scientific landscape. We built upon published protocols, vendor manuals, and prior structural biology literature, acknowledging openly where we borrowed methods and where we innovated. For example, we adapted protocols for K. phaffi expression from Thermo Fisher manuals while adding our own design of codon optimized IF variants from multiple species (see Experiments page). This integrity in acknowledging prior work ensures that our project is transparent, reproducible, and positioned as a contribution to collective knowledge rather than as an isolated effort.
While our initial roadmap included higher‑resolution binding analysis by isothermal titration calorimetry (ITC) and elemental cobalt quantification via ICP‑MS, the ITC instrument was unavailable due to maintenance, and ICP‑MS access was not possible within our timeframe. Rather than attempting unsafe or unvalidated workarounds, we substituted these methods with a UV‑Vis spectrophotometric binding measurement (see Measurement 1) and microwave plasma atomic emission spectroscopy (MP‑AES), openly reporting cases where signals were below detection limits (see Measurement 4). Similarly, although structural crystallography was part of our long‑term vision, we documented a detailed crystallization protocol for future teams instead of rushing into incomplete trials (see Measurement 3).
At every technical stage from cloning (Experiments 1-7) to safe expression in K. phaffi (Experiments 9-10) and purification with Ni-NTA resin under controlled conditions (Experiment 12), we followed vendor safety guidelines, avoided hazardous shortcuts, and disclosed when contamination or low yields limited downstream analysis. By acknowledging these constraints in our wiki and lab notebook, and framing alternative assays that could be safely performed in our lab, we maintained integrity and transparency. Learn more about our efforts towards safety and risk management from our project safety form.
Finally, by framing our solution in the “big picture” context of improving B12 absorption for patients with intrinsic factor deficiencies, we remain both humble and creative. Humble in admitting that we cannot solve every problem at once, but creative in offering a realistic, safe, and scientifically sound path forward. This integrity in how we approached limitations is a hallmark of responsible synthetic biology and demonstrates our commitment to the values that iGEM encourages teams to embody.
