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Single Molecule Investigations in Living E. coli (SMILE)
Date du début: 1 avr. 2014, Date de fin: 31 mars 2019 PROJET  TERMINÉ 

The overall ambition is to make intracellular measurements that bridge the gap between quantitative physical models and biological observations, in order to identify and resolve inconsistencies in our current understanding of life at the molecular level.We are particularly interested in answering fundamental questions about how the molecules that regulate gene expression and DNA replication operate in the intracellular environment. These questions include how transcription factors and RNA polymerases have evolved to rapidly find and bind their specific binding sites on chromosomal DNA, how the initiation of DNA replication is coordinated with cell growth, how cells can filter out leakage during replication of repressed genes, and other questions related to how the physical limitations of molecular interactions constrain cellular regulation.Answering these questions requires better methods to study intracellular kinetics at high spatial and temporal resolution. For this purpose we will develop single-molecule methods to probe kinetics without perturbing the system. The project depends on development in five different fields (1) new labeling methods using unnatural amino acids, (2) new hardware solutions for single molecule tracking, (3) new microfluidic designs for controlled growth conditions and image analysis, (4) new computational tools to extract the maximal amount of information from many short single molecule trajectories and (5) accurate ways of computing synthetic microscopy images.In addressing the specific questions stated above the experiments are necessarily combined with biophysical modeling that makes it possible to tell if the experimental results are expected from the previously known interactions and physical limitations due to for example stochasticity, slow diffusion and confined geometries. Overall the project is unique in its ambition to make a physically consistent quantitative description of central processes of life at the molecular level.