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@ARTICLE{Kipper:120470,
      author       = {K. Kipper and E. G. Lundius and V. Ćurić and I. Nikić
                      and M. Wiessler$^*$ and E. A. Lemke and J. Elf},
      title        = {{A}pplication of {N}oncanonical {A}mino {A}cids for
                      {P}rotein {L}abeling in a {G}enomically {R}ecoded
                      {E}scherichia coli.},
      journal      = {ACS synthetic biology},
      volume       = {6},
      number       = {2},
      issn         = {2161-5063},
      address      = {Washington, DC},
      publisher    = {ACS},
      reportid     = {DKFZ-2017-00899},
      pages        = {233 - 255},
      year         = {2017},
      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.},
      cin          = {E020},
      ddc          = {540},
      cid          = {I:(DE-He78)E020-20160331},
      pnm          = {315 - Imaging and radiooncology (POF3-315)},
      pid          = {G:(DE-HGF)POF3-315},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:27775882},
      doi          = {10.1021/acssynbio.6b00138},
      url          = {https://inrepo02.dkfz.de/record/120470},
}