Investigating DNA Replication in Escherichia Coli on the Single Cell Level Utilizing Microfluidics and Single-Molecule Fluorescence Microscopy

Abstract

Chromosomal DNA replication is a highly dynamic and complex process. Multiple enzymes work closely together to successfully synthesize new DNA before cell division occurs. The current understanding of this process is largely based on ensemble averaging experiments. These types of experiments, while powerful, can obscure the events that occur between individual molecules. We build on the current understanding of DNA replication by studying this process on the single-cell level. We use a combination of quantitative in vivo single-molecule fluorescence microscopy and microfluidics to probe the dynamics of specific molecules involved during replication inside individual living E. coli cells. In particular, we employ custom-built microfluidic channels to immobilize the bacteria in such a manner as to study individual cells for multiple generations. This not only allows us to image cells without fixation, but it also provides the added benefit of obtaining a large amount of information regarding a specific cell in a single experiment, including repeated observation of the different phases of its cell cycle. Here we describe the techniques involved in these experiments as well as the implications of our data for our understanding of the replication process.