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Updated: Jan 9, 2026
Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion
Published on: February 10, 2023
Optimization of the genetic code expansion technology for intracellular labelling and single-molecule tracking of
Filip Ilievski1,2, Linnea Wikström1, Anneli Borg1
1Department of Cell and Molecular Biology, Uppsala University Uppsala Sweden m.johansson@icm.uu.se.
Abstract:
Single-molecule tracking (SMT) is a powerful tool for real-time studies of protein interactions in living cells. Dye-labelled SNAP-tag and HaloTag self-labelling proteins have simplified SMT significantly, due to their superior photophysical properties compared to fluorescent proteins. However, due to their size, fusion of these tags to a protein of interest often results in loss of protein function. We introduce FLORENCE - a universal labelling method for SMT, based on genetic code expansion (GCE). We overcome significant caveats related to re-coded strains, vectors, and dyes and report successful tracking of site-specifically intracellularly labelled proteins in genomically re-coded E. coli. Our findings establish a robust in vivo protein-labelling strategy, expanding the capabilities of SMT as a method to study the dynamics of proteins in living cells. Moreover, we observe that the strain-promoted azide-alkyne click-chemistry reaction occurs as fast as 30 min in live E. coli cells and can be used as a robust labelling reaction.
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