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SMILE Report Summary

Project ID: 616047
Funded under: FP7-IDEAS-ERC
Country: Sweden

Mid-Term Report Summary - SMILE (Single Molecule Investigations in Living E. coli)

Historically, the interior of the biological cell has been a very mysterious place and most of what we have learned about biological processes has been deduced from experiments with purified components in test tubes. With increased microscopy power it eventually became possible to get a snapshot of this tiny biological factory and during the last decade, technological progress has enabled the study of biochemical reactions as they happen, inside the living organism, one molecule at the time. If it was the technological advances that allowed us to broaden, or rather lessen, our horizon or if is it our urge to peer deeper into the science of life that has driven the technology forward is hard to tell, but it is clear that the challenges of understanding life at the molecular level require methods with spatial precision in the nm range and time resolution in the order of milliseconds. In this project we have made progress towards increased resolution in time and space by developing new imaging modalities. In another approach we utilize the fact that fluorophores emit polarized light and when the molecules rotate, the corresponding polarization changes can be recorded with very high time resolution.
We use these techniques to study basic biological phenomena such as replication of the genome and gene regulation. The fundamentality of these processes makes them at the same time scientifically interesting and notoriously hard to study as interventions inevitably have severe consequences, both in terms of cell viability and secondary effects on the system. This complexity makes it necessary to work with computational models. The models are used to make predictions of what effect different perturbations will have on the system, predictions that we can subsequently test in the experimental set-ups to determine if the models are plausible. Using this approach, we have characterized how the replication and division cycles are coupled in individual E. coli cells, which in turn made it possible to sort out the enduring issue about the origin of variability in cell sizes and generation times. Another longstanding question investigated in the lab is how molecules that regulate gene expression can rapidly find their specific target site among the myriads of similar sequences on the DNA. The combination of computational and experimental methods has given us an increasingly clear picture about this process where the searching protein uses a combination of 3D diffusion and 1D spiraling along the DNA to speed up the search process.
One of our biggest challenges lies in visualizing the different components of the biological systems without disturbing their function. In this project we have developed new techniques of protein and RNA labeling with small, bright molecules by hijacking different molecular processes in the cell. For example it is possible to insert non-canonical amino acids into proteins and use these special residues as docking sites for small molecular dyes. The method works well for labeling of extracellular proteins but there is still work to be done to make the dyes efficiently cross the cell membrane.

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