NDLI: Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation

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Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation

Content Provider	PubMed Central
Author	Marko, Sustarsic Anne, Plochowietz Aigrain, Louise Yuzenkova, Yulia Zenkin, Nikolay Kapanidis, Achillefs
Abstract	Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities and mechanisms, and to validating in vitro characterization. Improvements in labeling and imaging technologies are starting to allow such in vivo studies; however, a number of technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores to be introduced at high efficiency into live E. coli (Crawford et al. in Biophys J 105 (11):2439–2450, 2013). Here, we address important challenges related to internalization of proteins, and optimize our method in terms of (1) electroporation buffer conditions; (2) removal of dye contaminants from stock protein samples; and (3) removal of non-internalized molecules from cell suspension after electroporation. We illustrate the usability of the optimized protocol by demonstrating high-efficiency internalization of a 10-kDa protein, the ω subunit of RNA polymerase. Provided that suggested control experiments are carried out, any fluorescently labeled protein of up to 60 kDa could be internalized using our method. Further, we probe the effect of electroporation voltage on internalization efficiency and cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Förster resonance energy transfer.
Related Links	http://dx.doi.org/10.1007/s00418-014-1213-2
Ending Page	124
Page Count	12
Starting Page	113
File Format	PDF
ISSN	1432119X
e-ISSN	1432119X
Journal	Histochemistry and Cell Biology
Issue Number	1
Volume Number	142
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2014-01-01
Access Restriction	Open
Rights Holder	Springer Berlin Heidelberg
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Histology Molecular Biology Medical Laboratory Technology

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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