Detection of in vivo Protein Interactions in All Bacterial Compartments by Förster Resonance Energy Transfer with the Superfolder mTurquoise2 ox-mNeongreen FRET Pair.

This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in all compartments of Escherichia coli by Förster Resonance Energy Transfer (FRET) using the Superfolder mTurquoise2 ox-mNeonGreen FRET pair (sfTq2^ox-mNG). This FRET pair has more than twice the detection range for FRET interaction studies in the cytoplasm or periplasm of E. coli compared to other pairs to date. These protein-interaction studies can be performed in vivo because fluorescent proteins can be genetically encoded as fusions to proteins of interest and expressed in the cell. sfTq2^ox and mNG fluorescent protein fusions are co-expressed in bacterial cells and the fluorescence emission spectra are measured. By also measuring reference spectra for the background, sfTq2^ox-only and mNG-only samples, expected emission spectra can be calculated. Sensitized emission for mNG above the expected spectrum can be attributed to FRET and quantified by spectral unmixing. This bio-protocol discusses the sfTq2^ox-mNG FRET pair and provides a practical guide in preparing the protein fusions, setting up and running the FRET experiments, measuring fluorescence spectra and gives the tools to analyze the collected data.