Single-molecule imaging in live cell to study gene geography and the role of DNA biophysics in regulating genes

Your project will address the key question of what is the importance of the physical location of genes in determining their state of activity. We will aim to address this question by studying the effects of bacterial chromosomal gene order on spatiotemporal expression in the context of folded, native genomes. You will use single-molecule bioimaging and computational analysis to determine the output from genes are their location is changed, and how this is affect to whether or not the cell is stressed. This will give a unique insight into how the very local state of DNA topology affects whether or not genes in live cells are switched on or off. Specifically:

1. You will characterize the spatiotemporal localization of new E. coli bacterial strains which expressing genomic fusions to a key enzyme DNA gyrase which relieves DNA torsional stress. These are constructed to use a nucleoid associated protein Fis as a marker for DNA supercoiling inside each cell.

2. You will use super-resolution millisecond Slimfield microscopy which is sensitive at a level of single-molecule detection to determinate the dynamic pattern of spatial localization and stoichiometry of these gyrase subunit proteins GyrA and GyrB to nanoscale precision in single live E. coli cells.

3. You will help to construct under expert teaching several dual-colour fluorescent protein fusion constructs integrated into the E. coli genetic code to pairs of DNA gyrase subunits, and also to Fis, and to a range of different established genome loci markers.

4. You will use nanoscale co-localization microscopy, real-time FRET, and FRAP/FLIP to determine the dynamic moelcular interactions, kinetics and molecular turnover of gyrase components in live cells under a range of bengin and stressed conditions including the appliation of gyrase targetting antibiotics.