Integrated Human Practices


Design Considerations


From the survey and all interviews, we learned that lactose intolerance is a very common comorbidity alongside celiac disease. Of the 10 respondents who answered this optional question about health and personal context, 5 reported being lactose intolerant and/or allergic to dairy products.

In the early stages of our design, our focus was on finding a promoter that would effectively drive expression of the enzyme or peptide responsible for gluten cleavage or masking. The lac promoter was an obvious choice for initial testing, as it can be easily induced in the lab with lactose or IPTG [1].

lac operon
Figure 1. Regulation of the lac operon. When lactose is absent (top), a repressor protein binds to the operator, blocking RNA polymerase and preventing transcription. When lactose is present (bottom), allolactose binds to the repressor, removing it from the operator and allowing transcription of the lacZ, lacY, and lacA genes. (from [2])

However, ethical and safety concerns quickly arose. Using a lactose-inducible promoter could potentially exclude individuals with other dietary restrictions, whether for medical, religious, or ethical (e.g., vegan) reasons. More importantly, we also discussed the risks of a constitutively active promoter. Some argued that a constantly active promoter might overburden or even damage the gut environment, while others noted that if the probiotic is unable to colonize the gut for long periods, its short lifespan would naturally limit potential harm—though this would require more frequent dosing, as seen with several commercial probiotics.

These questions pushed us to explore alternative systems. While looking through the iGEM Registry and later the 2025 distribution plates, we found promoters from the Anderson collection that use arabinose as an inducer. Arabinose is a plant-derived sugar naturally present in fruits and vegetables. This offers a promising option, as it aligns better with the dietary constraints and preferences of individuals with gluten intolerance.

Although the arabinose system appeared to be a strong solution, we also considered more project-specific ideas:

  • Gluten-inducible promoter (positive inducible): One idea was to design a promoter activated by the presence of gluten itself. While conceptually targeted, this approach would not work in practice inside the gut. The body’s immune system detects gluten rapidly—likely much faster than our engineered promoter could respond—meaning the immune response would be triggered before our system had the chance to act.
  • Substrate-inhibited promoter (negative repressible): Another idea was to use a system where the enzyme product (or substrate byproduct) would feed back to inhibit the promoter. This design had the advantage of limiting overproduction by creating a natural feedback loop—expression would continue until the product concentration reached a certain threshold, at which point transcription would shut down. However, this system would require significant testing to determine appropriate concentration thresholds and fine-tune sensitivity, since imbalances could result in under- or over-expression in vivo.

For the purposes of our current proof-of-concept and lab testing, we continued with the lac promoter, as it is straightforward and reliable in an experimental context. Nevertheless, we recognize that identifying a safer and more inclusive promoter system—such as one induced by arabinose, or potentially a feedback-regulated system—will be essential for any future development of this project.

Impact on Testing and Validation


The interview conducted with Dr. Amélie Therrien, as well as our survey, also permitted some adjustments of our testing conditions in the following ways:

  • We learned through our interview with Dr. Therrien that the threshold for a product to be considered gluten-free, as regulated by the Food and Drug Regulations (FDR), is 20 ppm [3], as it is the gluten concentration above which most patients begin to experience symptoms related to gluten exposure. However, some patients may still experience symptoms at lower concentrations of gluten.
  • The responses to our survey showed that most people with celiac disease experience symptoms in a range of 0–3 hours after exposure to gluten (check out the insights of our survey)
Taken together, these findings guided our experimental design by defining more realistic and clinically relevant testing ranges. When developing our colorimetric assay, we used these insights to determine the gluten concentrations and time intervals at which to evaluate enzyme activity, as described in the experiments section.

References


  1. Sanganeria, T., & Bordoni, B. (2022, October 17). Genetics, Inducible Operon. PubMed; StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK564361/
  2. Khan Academy. (2021). The Lac Operon. Khan Academy. https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/regulation-of-gene-expression-and-cell-specialization/a/the-lac-operon
  3. Canadian Food Inspection Agency. (2024). Compliance and enforcement of gluten-free claims - inspection.canada.ca. Canada.ca. https://inspection.canada.ca/en/food-labels/labelling/industry/allergens-and-gluten/gluten-free-claims