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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.

Protein synthesis is one of the central processes of life. With recent single-molecule tracking techniques, detailed information about protein synthesis dynamics has been extracted from living cells. Still, there are conflicting results and very little knowledge about the dynamic behaviour of elongation factors because of the lack of fluorescent labels that preserve their native function. In the present study, we combine single-molecule tracking with the genetic code expansion (GCE) technology to directly observe ribosome binding dynamics of translation elongation factor EF-G in growing E. coli cells. We show that EF-G cannot be functionally labelled with the commonly used HaloTag. Instead, we utilize bio-orthogonal click-chemistry to fluorescently label EF-G in an optimized re-coded E. coli. This system reports the first estimation of the ribosome-binding time of EF-G in vivo, contributing to measuring the kinetics of elongation under physiological conditions. These findings open a technological avenue for difficult-to-label molecules inside living cells.

Genomically recoded Escherichia coli (GRE) strains represent a valuable tool as model organisms for single-molecule tracking via click-labelling. These strains are constantly being engineered to incorporate non-canonical amino acids, enabling expanded chemical functionality. However, extensive genome-scale recoding frequently reduces their fitness and imposes metabolic re-wiring limiting the utility of GREs as practical model organisms for live-cell imaging in vivo. This study reports that previously optimized GRE6 was subjected to adaptive laboratory evolution (ALE) in an imaging-rich defined media (RDM) to recover fitness. The evolved strain displayed improved growth relative to the ancestral GRE6, and a wild-type-like growth was confirmed by single-cell analysis. Whole-genome sequencing revealed deletion of the initiation inhibitor ratA. ncAA incorporation was comparable or slightly improved relative to the common MG1655 strain. These results point out the importance of applying ALE in parallel to rational genome engineering in expanding the benefits of genomically re-coded organisms. Moreover, they point out the necessity for the development of improved vectors for essential translation factors for SMT.