Tracking Bacterial Chromosome Dynamics with Microfluidics-Based Live Cell Imaging
In bacteria, chromosomes are highly organized within the limited volume of the cell to form a nucleoid. Recent application of microscopy and chromosome conformation capture techniques have together provided a comprehensive understanding of the nature of this organization and the role of factors such as the structural maintenance of chromosomes (SMC) proteins in the establishment and maintenance of the same. In this chapter, we outline a microfluidics-based approach for live cell imaging of Escherichia coli chromosome dynamics in wild-type cells. This assay can be used to track the activity of the SMC complex, MukBEF, on DNA and assess the impact of perturbations such as DNA damage on chromosome organization and segregation.
Key wordsMicrofluidics Fluorescence microscopy Chromosome DNA damage SMC proteins MukBEF Escherichia coli
We thank Dr. Asha Mary Joseph and other lab members for comments on the manuscript, Dr. Sandler for sharing the strain with hupA-mCherry, and Dr. Reyes-Lamothe for the strain with mukE-mYPet. We would also like to acknowledge Merck for providing permission to use images from their user manual. AB is funded by the Tata Institute of Fundamental Research and a Career Development Award from the Human Frontier of Sciences Program.
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