Team:REC-CHENNAI/Project-Description
PROJECT DESCRIPTION
We propose an alternate delivery method for the transient transfection of the cargo. The chimeric Cell-Penetrating Peptide BP100-(KH)9 has a broad range of specificity, having been used to transfect plant cells and transform bacterial cells. We have designed a system for screening the successful transfection. We propose to validate our system in the context of RNA interference.
The shRNA like siRNA (referred to as siRNA from now on) targeting the mRNA is the biobrick designed by us. It is conjugated with a fluorophore and a quencher generating RNA beacon.
When we presented our system to Dr.D.Karunagaran, an eminent scientist and professor who works on the role of miRNAs in cancer apoptosis, he preferred that we chose some other gene for validating our idea to c-myc mRNA in HeLa cell line. So we decided to target Survivin mRNA owing to its high expression in cancer cell lines.
In the shRNA-like form of our biobrick, the fluorescence emitted by the fluorophore is quenched by the quencher. When this biobrick is transfected with CPP, the interfering RNA binds to its target i.e. the Survivin mRNA and unwinding of the strands takes place due to difference in the binding affinity which leads to the spatial separation of the fluorophore and the quencher. Thus fluorescence is unquenched and can be detected. The fluorescence thus emitted gives a preliminary confirmation of the binding and hence the silencing of gene expression. We confirm the same by the following experiments:
