Design and cloning of a system to visualize transcription in real time based on protein complementation
Protein complementation, or more precise protein-fragment complementation, is a method to study protein-protein interactions. Two proteins are tagged with incomplete fragments of a reporter protein (e.g. GFP). if the two proteins come close together for a possible interaction, the parts of the reporter protein form a functional reporter and can be measured. 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 protein complementation. The system should be designed for bakers yeast with a inducible promoter to start transcription as wanted.
Familiar with following basic laboratory work: PCR, primer design, transformation, ligation, restriction, homologous recombination, gel electrophoresis, plasmid preparation
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