Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli

Abstract

Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.

An inside look at a biofilm: Pseudomonas aeruginosa flagella bio-tracking

“…However, despite a consensus among researchers that flagella are not present during biofilm maturation but only in the dispersion stage, reports regarding this are still somewhat constradicting [10][11][12][13][14][15][16] . Therefore, it is necessary to To date, live-cell imaging can be obtained through different approaches, however genetic code expansion, the reassignment of codons and incorporation of an unnatural amino acid (Uaa) into proteins 17 , displays advantages over other methodologies, and is gaining increasing exposure and momentum [18][19][20][21] . Genetic code expansion systems are being constantly improved, expanded and adapted to a growing number of organisms [22][23][24][25] .…”

Visualizing the inner life of microbes: practices of multi-color single-molecule localization microscopy in microbiology

“…by immunofluorescence using anti-or nanobodies [36,37,39,40,50,51,53,56] (ii), by fluorescently labeled ligands, drug molecules or targeting proteins [15] (iii) or by aptamers (iv). Furthermore, the POI can be modified, either by a FP (v, PAmCherry ( pdb 3KCT)) [13,14,28,40,43,58,59] or by an enzyme/peptide tag (vi, SNAP ( pdb 3L00)) [21,30,35,36,38,39,41,56,63] or by insertion of an unnatural amino acid into the primary structure of the protein (vii), which after protein folding is coupled to a dye by a click chemistry reaction [64,65]. (c) Staining of native carbohydrates or lipids can be facilitated in a similar fashion as for proteins.…”

Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

“…The functions of these nsAAs range from biorthogonal tagging to photocrosslinking and to fluorescence, with broad experimental utility. To facilitate the further application of this technology and explain complications noted in previous work 21,22 , we firstly show that unlike in non-recoded E. coli [23][24][25] , nsAAs incorporate efficiently at amber stop codons in native B. subtilis genes. Secondly, the incorporation of photocrosslinking nsAAs allows demonstration of binding interactions of secreted proteins homologous to virulence factors.…”