DNA

Part:BBa_K3009030

Designed by: Carolin Ruckes   Group: iGEM19_Freiburg   (2019-10-19)


MJ tyrosyl synthetase

Group: Freiburg
Author: Alisa
Summary: The tyrosyl-tRNA -synthetase from M.jannashii and cognate tRNA pair are acting orthogonally to the endogenous translational machinery of E.coli. By binding and loading non-canonical amino acids onto the tRNA, the tyrosyl-tRNA -synthetase enables their incorporation into proteins. The tRNA is cognate towards the amber stop codon, which is suppressed by the non-canonical amino acid. [1]
Documentation:

Usage

The tyrosyl-tRNA-synthetase was engineered to recognize and load para-cyano-phenylalanine onto its cognate tRNA, which likewise has been mutated to be complementarity towards the amber stop codon. [1] The synthetase exhibits an unusually high substrate promiscuity. Being able to incorporate over 20 distinct non-canonical amino acids, with several D-amino acids among them, onto the tRNA. [2] Therefore, D-amino acids can be incorporated into peptides and proteins bearing amber stop codon mutations.

Biology

In nature, the tyrosyl-tRNA-synthetase catalyzes the bond between the tyrosyl-tRNA and tyrosine in the archaea Methanocaldococcus jannaschii. The tRNA can then bind to the complementary codon of a translated mRNA sequence and enable the incorporation of a tyrosine into the protein. [3]

Characterization

We employed the tyrosyl-tRNA-synthetase to incorporate D-phenylalanine into various amber-stop-codon-bearing superfolder GFP (sfGFP) mutants. Incorporation was demonstrated by quantifying the emitted fluorescence by the sfGFP protein.


Fig.1: Fluorescence signal restoration in C321.deltaA E.coli cells expressing sfGFPY66* and tyrsosyl-tRNA-ynthetase under a widefField microscope, 100X magnification and 475 nm excitation wavelength
Fig.2: Untransformed C321.deltaA cells under a widefield microscope, 100X magnification and 475 nm excitation wavelength.

Backbone: pULTRA Promoter: lacI, proK E.coli strain: C321.deltaA


References 

[1] Chatterjee, et. Al, (2013): A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. In: Biochemistry 52 (10), S. 1828–1837.

[2] Ma et. al (2015): Genetic incorporation of d -amino acids into green fluorescent protein based on polysubstrate specificity. In: RSC Adv. 5 (49), S. 39580–39586 [3] Italia et. al (2018): Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes. In: Cell chemical biology 25 (10), 1304-1312.e5.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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