Template:Heidelberg/collab/michigan

Collaboration with iGEM Team Michigan

The Michigan iGEM Team shares the fascination for the use of aptamers in synthetic biology. Their aim is to develop an aptamer based detection system for pathogen related proteins. To create a robust test strip, they implemented their sensor in a cell free expression system using a different approach for riboswitch design. The actual aptamer sensing the substances is a small DNA oligonucleotide which is bound to a trigger DNA oligo. On binding of the aptamer, the trigger is released and turns on a RNA toehold switch leading to the reporter expression. To further extend their system, they asked us for the development of new aptamer sequences to be implemented in their sensor system. We therefore worked on the computational creation of aptamer candidates for proteins and small molecules with our software that might be implemented into their system. Furthermore we discussed ways to implement new aptamers in their existing system. In this process we assessed whether the use of our JAWS software may enable them to fuse a variety of aptamers to a single toehold switch construct. In this process we sketched a potential way for switching larger toeholds with very small aptamers.

To be more precise, we included several of the kanamycin aptamers which we validated in our project work into the toehold switch they intended to use. The figure shows the two states of the toehold switch for one candidate we designed. On the left-hand side, the state with no kanamycin bound is depicted. The toehold-switch is inactive as the Ribosome Binding Site (RBS) is hidden in a strong stem. Upon binding of kanamycin, the regulatory construct that is fused to the aptamer and that keeps the toehold-switch in its inactive conformation (by not allowing base pairing between the regulatory construct and the toehold-switch) changes secondary structure. Thereby a previously existing strong stem unfolds and reveals a sequence complementary to the toehold-switch. As soon as this happens, the conformation on the right-hand side is formed and the RBS can be accessed.