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Genetic Encoding of Unnatural Amino Acids in C. elegans

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Noncanonical Amino Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1728))

Abstract

Site-specific incorporation of unnatural amino acids (UAAs) has greatly expanded the toolkit available to study biological phenomena in single cells. However, to address questions involving complex cellular interactions such as development, ageing, and the functions of the nervous system it is often necessary to use multicellular model organisms. The nematode Caenorhabditis elegans was the first organism to have its genetic code expanded. Due to its small size, ease of cultivation, and excellent UAA incorporation efficiency, C. elegans makes an ideal model organism to apply UAAs as tools to investigate the functioning of multicellular systems.

Here, we describe methods to generate transgenic C. elegans capable of UAA incorporation, as well as how to deliver unnatural amino acids and test incorporation. Furthermore, we describe methods to uncage photosensitive unnatural amino acid derivatives.

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References

  1. Chin JW, Cropp TA, Anderson JC et al (2003) An expanded eukaryotic genetic code. Science 301:964–967. https://doi.org/10.1126/science.1084772

    Article  CAS  PubMed  Google Scholar 

  2. Wang L, Brock A, Herberich B, Schultz PG (2001) Expanding the genetic code of Escherichia coli. Science 292:498–500. https://doi.org/10.1126/science.1060077

    Article  CAS  PubMed  Google Scholar 

  3. Ye S, Huber T, Vogel R, Sakmar TP (2009) FTIR analysis of GPCR activation using azido probes. Nat Chem Biol 5:397–399. https://doi.org/10.1038/nchembio.167

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ye S, Zaitseva E, Caltabiano G et al (2010) Tracking G-protein-coupled receptor activation using genetically encoded infrared probes. Nature 464:1386–1389. https://doi.org/10.1038/nature08948

    Article  CAS  PubMed  Google Scholar 

  5. Li C, Wang G-F, Wang Y et al (2010) Protein (19)F NMR in Escherichia coli. J Am Chem Soc 132:321–327. https://doi.org/10.1021/ja907966n

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jackson JC, Hammill JT, Mehl RA (2007) Site-specific incorporation of a 19F-amino acid into proteins as an NMR probe for characterizing protein structure and reactivity. J Am Chem Soc 129:1160–1166. https://doi.org/10.1021/ja064661t

    Article  CAS  PubMed  Google Scholar 

  7. Mori H, Ito K (2006) Different modes of SecY–SecA interactions revealed by site-directed in vivo photo-cross-linking. Proc Natl Acad Sci 103:16159–16164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chin JW, Martin AB, King DS et al (2002) Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc Natl Acad Sci 99:11020–11024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Nikić I, Kang JH, Girona GE et al (2015) Labeling proteins on live mammalian cells using click chemistry. Nat Protocols 10:780–791. https://doi.org/10.1038/nprot.2015.045

    Article  PubMed  Google Scholar 

  10. Plass T, Milles S, Koehler C et al (2012) Amino acids for Diels-Alder reactions in living cells. Angew Chem Int Ed 51:4166–4170. https://doi.org/10.1002/anie.201108231

    Article  CAS  Google Scholar 

  11. Lang K, Davis L, Torres-Kolbus J et al (2012) Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat Chem 4:298–304. https://doi.org/10.1038/nchem.1250

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lang K, Davis L, Wallace S et al (2012) Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions. J Am Chem Soc 134:10317–10320. https://doi.org/10.1021/ja302832g

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Uttamapinant C, Howe JD, Lang K et al (2015) Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins. J Am Chem Soc 137:4602–4605. https://doi.org/10.1021/ja512838z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gautier A, Nguyen DP, Lusic H et al (2010) Genetically encoded Photocontrol of protein localization in mammalian cells. J Am Chem Soc 132:4086–4088. https://doi.org/10.1021/ja910688s

    Article  CAS  PubMed  Google Scholar 

  15. Gautier A, Deiters A, Chin JW (2011) Light-activated kinases enable temporal dissection of signaling networks in living cells. J Am Chem Soc 133:2124–2127. https://doi.org/10.1021/ja1109979

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nguyen DP, Mahesh M, Elsasser SJ et al (2014) Genetic encoding of photocaged cysteine allows photoactivation of TEV protease in live mammalian cells. J Am Chem Soc 136:2240–2243. https://doi.org/10.1021/ja412191m

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Luo J, Arbely E, Zhang J et al (2016) Genetically encoded optical activation of DNA recombination in human cells. Chem Commun 52:8529–8532. https://doi.org/10.1039/C6CC03934K

    Article  CAS  Google Scholar 

  18. Greiss S, Chin JW (2011) Expanding the genetic code of an animal. J Am Chem Soc 133:14196–14199. https://doi.org/10.1021/ja2054034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Parrish AR, She X, Xiang Z et al (2012) Expanding the genetic code of Caenorhabditis elegans using bacterial aminoacyl-tRNA synthetase/tRNA pairs. ACS Chem Biol 7:1292–1302. https://doi.org/10.1021/cb200542j

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Radman I, Greiss S, Chin JW (2013) Efficient and rapid C. elegans transgenesis by bombardment and hygromycin B selection. PLoS One 8:e76019. https://doi.org/10.1371/journal.pone.0076019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lammers C, Hahn LE, Neumann H (2014) Optimized plasmid systems for the incorporation of multiple different unnatural amino acids by evolved orthogonal ribosomes. Chembiochem 15(12):1800–1804. https://doi.org/10.1002/cbic.201402033

    Article  CAS  PubMed  Google Scholar 

  22. Schmied WH, Elsasser SJ, Uttamapinant C, Chin JW (2014) Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered eRF1. J Am Chem Soc 136:15577–15583. https://doi.org/10.1021/ja5069728

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bianco A, Townsley FM, Greiss S et al (2012) Expanding the genetic code of Drosophila melanogaster. Nat Chem Biol 8:748–750. https://doi.org/10.1038/nchembio.1043

    Article  CAS  PubMed  Google Scholar 

  24. Lyssenko NN, Hanna-Rose W, Schlegel RA (2007) Cognate putative nuclear localization signal effects strong nuclear localization of a GFP reporter and facilitates gene expression studies in Caenorhabditis elegans. BioTechniques 43:596. 598, 560

    Article  CAS  PubMed  Google Scholar 

  25. Kervestin S, Jacobson A (2012) NMD: a multifaceted response to premature translational termination. Nat Rev Mol Cell Biol 13:700–712. https://doi.org/10.1038/nrm3454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Pulak R, Anderson P (1993) mRNA surveillance by the Caenorhabditis elegans smg genes. Genes Dev 7:1885–1897

    Article  CAS  PubMed  Google Scholar 

  27. Longman D, Plasterk RHA, Johnstone IL, Cáceres JF (2007) Mechanistic insights and identification of two novel factors in the C. elegans NMD pathway. Genes Dev 21:1075–1085. https://doi.org/10.1101/gad.417707

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Centre for Integrative Physiology, University of Edinburgh, Edinburgh, UK

    Lloyd Davis & Sebastian Greiss

Authors
  1. Lloyd Davis
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  2. Sebastian Greiss
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Corresponding author

Correspondence to Sebastian Greiss .

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Editors and Affiliations

  1. Structural and Computational Biology Unit & Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany

    Edward A. Lemke

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Davis, L., Greiss, S. (2018). Genetic Encoding of Unnatural Amino Acids in C. elegans . In: Lemke, E. (eds) Noncanonical Amino Acids. Methods in Molecular Biology, vol 1728. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7574-7_24

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  • DOI: https://doi.org/10.1007/978-1-4939-7574-7_24

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7573-0

  • Online ISBN: 978-1-4939-7574-7

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