Date : 09/06/2011

Internship proposal for : Master 1

Laboratory
Laboratoire de Physique Statistique de l'ENS UMR CNRS 8550
Ecole Normale Superieure, 24 rue Lhomond 75005 Paris
Director : Mr Eric PEREZ
Main discipline : Biophysics
http://www.lps.ens.fr/-Surfaces-moleculaires-organisees-

Mentor
Wladimir URBACH
email : This e-mail address is being protected from spam bots, you need JavaScript enabled to view it
phone : +33 6 88 58 09 78

Subjects
1.: Efflux pump production
2.: in vitro reconstituion
3.: lipid vesicles

Tools-Methodologies
1.: Light scattering (DQLS),
2.: Photo bleaching (FRAPP)
3.: Micromanipulation vesicles & confocal microscopy

Summary of lab's interests

Biomembrane dynamics : adhesion, fusion and protein transport, from the molecular to the cell level, force measuring techniques coupled to optical tools live or biomimetic systems. The techniques we use are micropipet micromanipulation (Biomembrane Force Probe, cell adhesion, vesicle adhesion), Surface Force Apparatus, coupled to optical tools such as FRAPP, TIRF and confocal microscopy.

Summary of project

Bacteria control their intracellular concentration of various substrates (antibiotics, heavy metals) by organizing their expulsion through efflux pumps the mechanism of which is unknown. These pumps, inserted into the bacterial membrane, can induce bacterial resistance to antibiotics. Understanding how they work would determine the way to block them and thus reduce the bacterial resistance. For gram-negative bacteria, these pumps are usually made of three parts: one in the outer membrane, another one in the inner membrane, and a third one located between the two membranes of the bacteria. The project aims to reconstitute a specific pump as a whole, within an artificial double bilayer system in order to study its function. We have previously shown that the model system is biologically relevant by measuring the activities of several trans-membrane proteins in an identical system. The production of all the three components of the pump is now under control. We first want to perform measurements on individual links between the different pump components to understand the stability of the whole pump. Afterwards the pump will be reconstituted in vitro by inserting the different pump components into the lipid membranes of vesicles. The study of its function will be performed by observing the crossing of fluorescent molecules from one vesicle to another.