Team:Strasbourg/Description

iGEM

Project Inspiration



Looks delicious, right? Unfortunately,
1 to 3% of people would face a severe
reaction if they ate those deserts.
The cause? The presence of peanuts,
which is often invisible in many meals.

We all know people who refuse to taste a cake or who are stressed when eating out because of allergies. They have to rely on labels and waiters to avoid allergic reactions, but sometimes it is not enough. We wanted to offer them a simple and reliable test to remove this stress from their shoulders.

The first problem we faced was to find a receptor capable of recognizing an allergen in a specific and quickly interchangeable way in order to be able to detect a wide range of allergens. The second one was to identify a signal transduction pathway capable of transmitting information about the presence of the allergen and converting it into an analyzable signal.

As Strasbourg is a research center on RNA, we were prone to work with this molecule, and, thanks to Jorg Hartig, we realized that aptazymes fulfilled our conditions. These small RNAs allow the specific recognition of an increasing number of molecules and possess an auto-catalyzing domain. We still needed a platform to visualize the interaction ligand/aptamer.



Project Description

AptaTest is based on a triple hybrid system implemented in E. coli. Flexibility is provided thanks to an aptazyme which can be interchanged for specific allergens.

We designed two constructs to be able to select the best one. In both, an aptazyme links an MS2 protein to a PP7 protein.

In the first construct, MS2 is linked to a domain recognizing a promoter upstream of a reporter gene repressor whereas PP7 is linked to the RNA polymerase, thus recruiting it. If the allergen is present in the food, the aptazyme will undergo cleavage which will block the RNA polymerase recruitment.

In the second construct, MS2 and PP7 are linked to LexA DNA binding domains which themselves bind to a LexA operator. When both are linked to the operator, it inhibits the transcription of a reporter gene. When the aptazyme is cleaved, it deactivates this repression, allowing the expression of the reporter.

We established a proof-of-concept and a prototype that are presented in the other tabs. We hope that AptaTest will improve allergic people life and that they will be able to eat as much cake as they want without any severe reaction!



References

  1. Berry, K. E., Hochschild, A. 2018. A bacterial three-hybrid assay detects Escherichia coli Hfq-sRNA interactions in vivo. Nucleic Acids Research 46.
  2. Daines, D. A., Granger-Schnarr, M., Dimitrova, M., Silver, R. P. 2002. Use of LexA-based system to identify protein-protein interactions in vivo. Methods Enzymol 358:153–161.
  3. Daines, D. A., Silver, R. P. 2000. Evidence for multimerization of Neu proteins involved in polysialic acid synthesis in Escherichia coli K1 using improved LexA-based vectors. J Bacteriol 182:5267–5270.
  4. Felletti, M., Stifel, J., Wurmthaler, L. A., Geiger, S., Hartig, J. S. 2016. Twister ribozymes as highly versatile expression platforms for artificial riboswitches. Nature Communications 7.
  5. Stifel, J., Spöring, M., Hartig, J. S. 2019. Expanding the toolbox of synthetic riboswitches with guanine-dependent aptazymes. Synthetic Biology 4.