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Directed evolution of RNA in microdroplets

Research unit

UPR 9002 - Architecture et réactivité de l'ARN (IBMC)
15, rue Rene Descartes 67084 - Strasbourg Cedex

Group

Name: Réseaux de reconnaissances biomoléculaires

Group leader: WESTHOF Eric - e.westhof@ibmc-cnrs.unistra.fr

Group leader's phone: 0388417046

Website: Visit website

Group organization:
- Chercheurs: 7
- ITA: 2
- Doctorants: 1
- Post-Docs: 1
- Autres: 0

Publications of the team linked to the topic (3 last years):
1) Fallah-Araghi, A., Baret, J.C., Ryckelynck, M., and Griffiths, A.D. (2012). A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution. Lab on a chip 12, 882-891.
2) Najah, M., Griffiths, A.D., and Ryckelynck, M. (2012). Teaching single-cell digital analysis using droplet-based microfluidics. Analytical chemistry 84, 1202-1209.
3) Cattenoz, P.B., Taft, R.J., Westhof, E., and Mattick, J.S. (2013). Transcriptome-wide identification of A > I RNA editing sites by inosine specific cleavage. RNA 19, 257-270.

About PhD

PhD Director: WESTHOF Eric - e.westhof@ibmc-cnrs.unistra.fr

Phone: 0388417046

Junior advisor: RYCKELYNCK Michael

Co-tutely: non

Co-Director: non

About PhD topic :

Title: Directed evolution of RNA in microdroplets

Project: This PhD project will aim at obtaining aptamers by directed evolution in microdroplets generated and handled using microfluidics technology. In a first step, the work initiated in the group on the improvement of fluorescence properties of Spinach RNA, an aptamer that specifically recognize a cofactor (DFHBI) and form a fluorescent complex, will be pursued. A new in vitro recombination approach will be used to generate large sequence and structural space, with the goal of multiplying the number of DFHBI binding sites while limiting size increase. After having sequenced evolved molecules, the most represented variants will be produced and purified, their fluorescence properties characterized and their secondary structure determined through chemical probing and bioinformatics structure predictions.
       The performance of the improved Spinach (iSpinach) will be evaluated in vivo using bacteria. The molecule will be expressed in E. coli alone, inserted in a stabilizing structure or in fusion with a messenger RNA and the bacteria fluorescence will be monitored in time-lapse experiments using our imaging platform. Once the system and its sensibility will have been validated, the new RNA motif will be transferred into pathogenic bacteria (e.g. S. aureus, L. monocytogenes…) in fusion with messenger or regulatory RNAs allowing for monitoring their expression along the time. This part of the project will be performed through collaborations with groups having expertise with those bacteria, especially Pascal Romby's one.
       The last part of this project will consist in the use of Spinach (or iSpinach) as fluorogenic reporter in experiments aiming at obtaining, through in vitro evolution experiments, news riboswitches controlling transcription. To do so, libraries will be generated starting from a known riboswitch in fusion with Spinach. Negative and positive selection cycles will be performed in microdroplets (with and without the metabolite that the riboswitch should recognize) and will allow for isolating new riboswitches that will find direct applications in synthetic biology.

Wished skills: Basics of organic chemistry, molecular biology, biochemistry

Expertises which will be acquired during the training: Microfluidics, digital biology, imaging, RNA structural biology