In Vivo Investigation of DNA Replication in Escherichia Coli using Single-Molecule Fluorescence Microscopy

Abstract

Our research involves investigating the process of DNA replication in living cells. More specifically the dynamics of the replisome components in living Escherichia coli cells during the replication termination process. Our current knowledge of the replisome dynamics, and the termination process, has been obtained mainly from in vitro experiments that were not done on the single molecule level. However, the natural environment of the cell is considerably different from that of in vitro solutions. These differences can have a significant influence on how certain proteins function in a cell. Interestingly, it is now possible to monitor single-molecule processes using fluorescence microscopy inside genetically modified living cells, by tagging their native proteins with fluorescent probes. In our research we employ single-molecule fluorescence microscopy to track individual replisomes with other replication-related proteins, and investigate their dynamics during the process of DNA replication in living bacterial cells. The single-molecule sensitivity can add to our understanding by providing further insights into processes that are hidden by ensemble-averaging techniques. Here we describe the microscopy techniques, the micro-fluidic devices and the various gene manipulation techniques utilized in researching the above mentioned molecules.View Large Image | View Hi-Res Image | Download PowerPoint Slide