We had the pleasure of collaborating with the Boston University Wetlab iGEM team!
One of the many factors we considered when choosing proteins for our system included its modularity, and having the ability to add additional domains. After much consideration, we decided on using Ms2 to build upon current mechanisms to control alternative splicing (i.e. SSOs). With a gRNA sequence attached to the Ms2 protein, not only is Ms2 able to bind to the target RNA molecule, but the presence of the Ms2 can stabilize and prevent the degradation of the RNA sequence before it gets the chance to bind. Furthermore, there lies the possibility of adding more domains to the Ms2 protein to increase the chances that the RNA won't get degraded and will bind to the target.
In theory, the robustness and modularity of Ms2 should be helpful in transporting RNA. To test this idea, we sought a collaboration with the BU Wetlab iGEM team whose project also relies on RNA.
In order to activate the toe hold switches, and therefore activate gene expression, there must be a "Trigger RNA" present to bind the hair pin loop on the "Switch RNA". This allows the ribosome binding sight to be accessible to the ribosome for translation. This process only works if the trigger RNA is present and not degraded.
This is where M2s comes in. We had Ms2 attached to the trigger sequence, where the trigger sequence in this case was an SSO. We hope that the presence of MS2 would decrease the likelihood of RNA degradation, while also allowing the possibility of adding additional domains to make RNA degradation even less likely.
How did our team benefit from this collaboration?The BU Wetlab team is using a cell-free system to test their toe hold switches. Since robustness was one of the criteria for choosing a sufficient ribosome binding protein (RBP) to control alternative splicing, we wanted to see how our Ms2 would fair in a cell-free system, and held up to our expectations.
We also scraped data from past iGEM websites and saved into a csv file.The file is initially used by the team to search for similar projects on CRISPR and RNA-targeting. It gathers various information, including title, abstract and content from project pages. The csv file was announced through the iGEM collaboration page, and we shared the file with multiple teams. We are glad that the file is used by Team Groningen for data visualization and analysis.