Skip to Main content Skip to Navigation
Theses

Observation de l'activité traductionnelle d'un ribosome unique par microscopie de fluorescence couplée à un système microfluidique

Abstract : This thesis recounts the conception and the realisation of an experiment about the study at the single molecule level of the prokaryotic ribosome activity. To do that, we use ribosomes and amino acids labeled with two fluorescent probes which emit at two different wavelengths. Thus, we are able to co-localize the ribosomes and the incorporated amino acids by single molecule total internal reflexion fluorescence microscopy. In order to count the number of incorporated amino acids, we studied, at the single molecule level, the photophysical properties of their probe : the organic fluorophore Bodipy FL. We used a buffer containing an oxygen scavenger and two chemical reagents, one oxydising and one reducting, to increase the life-time, by a factor of 25, and the fluorescence stability in reducing the blinking at low laser excitation intensities. For the single molecule ribosome study, we developped a versatile microfluidic cell. The surface chemistry was carefully elaborated to eliminate non specific interactions between the surface and the biological reagents, and to preserve the ribosome activity. We incorporated a specic tag in the ribosome to label it with a semiconductor nanocrystal (QD). The activity of the ribosome labeled with a QD was observed in bulk experiment, then at the single molecule level. The Bodipy FL labeled amino acid incorporation by single ribosomes was also observed. Finally, we present the first steps of the conception of an optical tweezer to determine messenger RNA cohesion forces.
Complete list of metadatas

Cited literature [27 references]  Display  Hide  Download

https://pastel.archives-ouvertes.fr/tel-00538401
Contributor : David Dulin <>
Submitted on : Tuesday, November 23, 2010 - 12:01:22 PM
Last modification on : Wednesday, September 16, 2020 - 5:43:40 PM
Long-term archiving on: : Friday, October 26, 2012 - 4:30:15 PM

Identifiers

  • HAL Id : tel-00538401, version 1

Citation

David Dulin. Observation de l'activité traductionnelle d'un ribosome unique par microscopie de fluorescence couplée à un système microfluidique. Physique [physics]. Université Paris Sud - Paris XI, 2009. Français. ⟨tel-00538401⟩

Share

Metrics

Record views

1034

Files downloads

277