Detection Of Protein Interactions In The Cytoplasm And Periplasm Of Escherichia Coli By Forster Resonance Energy Transfer
BIO-PROTOCOL(2018)
Abstract
This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in Escherichia coli by Forster Resonance Energy Transfer (FRET). The described assay allows for the previously impossible in vivo screening of periplasmic protein-protein interactions. In FRET, excitation of a donor fluorescent molecule results in the transfer of energy to an acceptor fluorescent molecule, which will then emit light if the distance between them is within the 1-10 nm range. Fluorescent proteins can be genetically encoded as fusions to proteins of interest and expressed in the cell and therefore FRET protein-protein interaction experiments can be performed in vivo. Donor and acceptor fluorescent protein fusions are constructed for bacterial proteins that are suspected to interact. These fusions are co-expressed in bacterial cells and the fluorescence emission spectra are measured by subsequently exciting the donor and the acceptor channel. A partial overlap between the emission spectrum of the donor and the excitation spectrum of the acceptor is a prerequisite for FRET. Donor excitation can cross-excite the acceptor for a known percentage even in the absence of FRET. By measuring reference spectra for the background, donor-only and acceptor-only samples, expected emission spectra can be calculated. Sensitized emission for the acceptor on top of the expected spectrum can be attributed to FRET and can be quantified by spectral unmixing.
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Key words
Bacteria,Cytoplasm,Periplasm,Protein interactions,FRET,mNeonGreen,SYFP2,mKO,mCherry
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