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Imaging of Single Dye-Labeled Chemotaxis Proteins in Live Bacteria Using Electroporation

  • Diana Di Paolo
  • Richard M. Berry
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1729)

Abstract

For the last 2 decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signaling in E. coli, including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical. For example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart. FPs may interfere with the native interactions of the protein, and their chromophores have low brightness and photostability, and fast photobleaching rates. Electroporation allows for internalization of purified CheY proteins labeled with organic dyes into E. coli cells in controllable concentrations. Using fluorescence video microscopy, it is possible to observe single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.

Keywords

Electroporation Single-molecule fluorescence microscopy Chemotaxis Bacterial motility 

Notes

Acknowledgments

We thank Prof. Achillefs N. Kapanidis for use of the Total Internal Reflection Fluorescence microscope. D.D.P. was supported by a UK EPSRC DTC studentship. BBSRC is thanked for continuous funding of the work in the Berry laboratory.

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Cambridge Healthcare ResearchSt John’s Innovation CentreCambridgeUK
  2. 2.Biological Physics Research Group, Clarendon Laboratory, Department of PhysicsUniversity of OxfordOxfordUK
  3. 3.Berry Group, Clarendon Laboratory, Department of PhysicsUniversity of OxfordOxfordUK

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