Design and cloning of a system to visualize transcription in real time based on FRET
Förster Resonance Energy Transfer (FRET) is a mechanism used in microscopy to quantify distances between objects, in most cases related proteins. One donor molecule (e.g. CFP) is excited with its specific wavelenght to fluorescence. If the acceptor molecule (e.g. YFP) is within 10 nm of distance to the donor, the emited light is absorbed by the acceptor for its own fluorescence. This can be utilized for RNA tagging methods. We work with the coating protein from Pseudomonas phage 7 (PP7) which binds to a specific RNA stem loop. Is the protein tagged with a fluorophore, one can follow transcriptional activity under the microscope in real time.
The combination of FRET technique with a RNA tagging technique results in a decrease of background signal for the acceptor. The challange is to design and clone a system based on PP7 and MS2 with a sequence of consecutively repeats of their stem loops to garantee the right distance for FRET. The system should be designed for bakers yeast with a inducible promoter to start transcription as wanted.
Familiar with the following basic laboratory work steps: PCR, primer design, transformation, ligation, restriction, homologous recombination, gel electrophoresis, plasmid preparation
Buxbaum, A. R., Haimovich, G., & Singer, R. H. (2014). In the right place at the right time: visualizing and understanding mRNA localization. Nature Reviews Molecular Cell Biology, 16(2), 95–109. doi:10.1038/nrm3918