Internalization and observation of fluorescent biomolecules in living microorganisms via electroporation.
The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisiæ, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.
Clarendon Laboratory, Department of Physics, University of Oxford; Wellcome Trust Sanger Institute, Genome Center.
Louise
L
Aigrain
Clarendon Laboratory, Department of Physics, University of Oxford.
Marko
M
Sustarsic
Clarendon Laboratory, Department of Physics, University of Oxford.
Robert
R
Crawford
Clarendon Laboratory, Department of Physics, University of Oxford.
Anne
A
Plochowietz
Clarendon Laboratory, Department of Physics, University of Oxford; a.kapanidis1@physics.ox.ac.uk.
Achillefs N
AN
Kapanidis
D004247:DNA / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics; D018274:Electroporation / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000032:analysis / Q000737:chemistry* / Q000493:pharmacokinetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism
D005456:Fluorescent Dyes; D004247:DNA