Diemer-T2

Design and cloning of a system to visualize transcription in real time based on protein complementation

Description

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.

Problem

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.

Requirements

Familiar with following basic laboratory work: PCR, primer design, transformation, ligation, restriction, homologous recombination, gel electrophoresis, plasmid preparation

Literature

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

Wu, B., Chen, J., & Singer, R. H. (2014). Background free imaging of single mRNAs in live cells using split fluorescent proteins. Scientific Reports, 4, 3615. oi:10.1038/srep03615

Contact

Jascha Diemer

Francois-Xavier Lehr