Contribution

Parts

We have developed a number of parts this year, for future teams to use in their projects:

The Advancement of Toehold Switches

Previously, toehold switches were only able to detect a maximum of two RNA strands at a time. Many teams in the past have submitted toehold switches with only one input such as the 2017 BATMAN project (Part:BBa_K2206000). The 2021 RIBOTOX project developed an AND-gate switch which can detect two RNAs simultaneously. The ability to detect two RNAs for one condition meant that luciferase was only produced when two trigger RNAs were present at sufficient concentrations which helped to increase the specificity. For other prevalent conditions however, this could prove problematic if multiple strands need to be detected as it is difficult to monitor and create the hardware to help detect a conglomerate of RNA. Furthermore, switches which induce a reporter protein in a CDS are unspecific. This is because the same miRNAs are upregulated in multiple conditions as extra-cellular microRNA networks are complex. We were able to scale up the design of past iGEM teams to produce systems that are able to detect several microRNAs. This exponentially increased specificity to the conditions we were testing for.

A new method to design toehold switches

Our team has created a software tool to generalise, generate and optimise our toehold switches for other iGEM teams to use in any desired scenario. It is designed to be very user-friendly and have integration with several different standards in bioinformatics to speed up the process of creating toehold switches. You can find more information about this on our Software page.

Issues with further development of Toehold Switches

Initially, our project tried to find the maximum generation of toehold switches that would feasibly work and react. To do this, prior to any software work, we tested a theoretical gen 5 toehold switch using 4 miRNA to compose the trigger structure by writing a script to vary the concentration of every strand in one of NUPCK’s tube ensembles. Once this was complete, we then wrote another script to find the combinations of concentrations of reactants that produced the maximum concentration of products. The maximum yield we found was that about 5% of switches would’ve activated, a percentage we deemed too low to continue perusing gen 5 switches or above.

Our Collaborative miRNA Database

We have started a Collaborative miRNA database, which we hope future teams will expand upon, and use in their future projects using toehold switches and more! You can find our full list below: