Team:Strasbourg/Demonstrate

iGEM

Proof of Concept

For the first time, the binding and catalytic activities of an aptazyme were used in a bacterial three-hybrid system to create a new detection molecular platform adapted to every molecule, thanks to the aptazyme versatility. In order to have an interaction between the aptazyme and the bacterial hybrid system containing MS2 and PP7 coat proteins, iGEM Strasbourg designed an aptazyme with MS2 and PP7 stem-loops (which have an high affinity to respectively MS2cp and PP7cp) at its extremities. Thanks to in vitro assay, we confirmed that modified aptazymes maintain their catalytic activity. We also investigated the compatibility of aptazymes with the bacterial three-hybrid system by in vivo ß-galactosidase assays. Our results prove that the theophylline aptazyme interacts with PP7 and MS2 coat proteins and that its cleavage prevents the reporter gene transcription. Therefore, an aptazyme combined with the bacterial triple hybrid system could represent a tool for the detection of allergens in food.

What to do next ?

In further investigations, the reporter strain needs to be selected for its ability to transport the allergen into the cytoplasm. We already found bacterial strains that interact with casein, lactose or translocate small peptides into the cytoplasm by ABC-transporters, but the transport of other allergens into cytoplasm needs to be tested. To improve our prototype, the used bacterial strain must be modified to add an internal control, which will indicate the functionality of the BH3 system. It could be a reporter gene managed by a constitutive promoter. This improvement will ensure the absence of false-negative results.

iGEM Strasbourg interested itself in existing aptamers for the most common allergens. We found five already validated DNA aptamers for allergens: Sialyllactose (Milk), Gluten (Gli4), Arachin (Peanut), LC6 and LC15 (Eggs). Now that the aptazyme has been designed, hypothetical sequences of aptazymes corresponding to each of the described allergens need to be analyzed in silico and by in vitro selection.

Moreover, genetic background influences aptazymes functionality (Xiang et al. 2019). Him and his team showed that the modification of the reporter gene can affect the functionality of aptazymes. The low levels of ß-galactosidase activity in the presence of theophylline ligand could be related to this property. Those observations enable and encourage the investigation of other reporter genes. For esthetic reasons and facility of data interpretation by customers during prototype use, we recommend to switch ß-galactosidase reporter to amiCP, blue chromoprotein obtained from the coral Acropora millepora and well-characterized by iGEM registry as BBa_K592009 part.

References

  1. Xiang, J. S., Kaplan, M., Dykstra, P., Hinks, M., McKeague, M., Smolke, C. D. 2019. Massively parallel RNA device engineering in mammalian cells with RNA-Seq. Nat Commun 10, 4327