Isolation and Characterization of Bacterial Nucleoids in Microfluidic Devices
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 wordsNucleoid isolation Microfluidics Nucleoid-associated proteins Molecular crowding
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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