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Isolation and Characterization of Bacterial Nucleoids in Microfluidic Devices

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1624)

Abstract

We report methods for isolation of Escherichia coli nucleoids in microfluidic devices, allowing characterization of nucleoids during a controlled in vivo to in vitro transition. Biochemically, nucleoids are isolated by gentle osmotic lysis, which minimally perturbs nucleoid-associated proteins (NAPs). Biophysically, nucleoids are isolated in microfluidic chambers, which mimic confinement within the cell, as well as facilitate diffusive buffer exchange around nucleoids without subjecting them to flow. These methods can be used to characterize interactions between NAPs and whole nucleoids, and to investigate nucleoid structure and dynamics in confinement. We present protocols for isolation, quantification, and perturbation of nucleoids in microfluidic confinement.

Key words

Nucleoid isolation Microfluidics Nucleoid-associated proteins Molecular crowding 

Notes

Acknowledgments

J.F.P. was supported by a Fannie and John Hertz Graduate Fellowship, and S.J. by Paul G. Allen Foundation, Pew Charitable Trusts, NSF CAREER, and NIH GM118565-01.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Center for Bits and AtomsMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Department of PhysicsMassachusetts Institute of TechnologyCambridgeUSA
  3. 3.UCSD Physics and Molecular BiologyLa JollaUSA

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