Optimization of the genetic code expansion technology for intracellular labelling and single-molecule tracking of proteins in genomically re-coded <i>E. coli</i>

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 <i>E. coli.</i> Our findings establish a robust <i>in vivo</i> 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 <i>E. coli</i> cells and can be used as a robust labelling reaction.