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Micro-manipulation de l'ADN Vers une visualisation directe par microscopie de fluorescence

Abstract : In this work, we present an apparatus designed to help unravel the molecular mechanism of a certain class of proteins. It combines the capabilities of a magnetic tweezers apparatus with those of a fluorescence microscope in TIRF mode. It has been designed to allow for mechanical manipulation and direct observation of DNA translocases at the single molecule level. We will first explain the design and testing of this setup. We will show that it does not only compare very well with the state of the art in magnetic tweezers or fluorescence microscope alone, but also that their combination lets us collect a new type of information. More specically, we studied the T7 RNAP polymerase and the FtsK motor from Escherichia coli . We will highlight the current questions arising concerning their mechanism of action, and propose a set of experiments to address them. Then we will explain why we had a lot of trouble obtaining the adequate fluorescent protein substrates, and how we believe we will solve this problem. We will finally analyze the results we obtained, either in bulk or magnetic tweezers experiments. They have unraveled new behaviours that we plan to use in new experiments on our combined MT/TIRF setup.
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https://tel.archives-ouvertes.fr/tel-00558689
Contributor : Adrien Meglio <>
Submitted on : Sunday, January 23, 2011 - 7:47:18 PM
Last modification on : Thursday, December 10, 2020 - 12:37:47 PM
Long-term archiving on: : Tuesday, November 6, 2012 - 12:05:19 PM

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  • HAL Id : tel-00558689, version 1

Citation

Adrien Meglio. Micro-manipulation de l'ADN Vers une visualisation directe par microscopie de fluorescence. Biophysique [physics.bio-ph]. Université Pierre et Marie Curie - Paris VI, 2010. Français. ⟨tel-00558689⟩

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