Part:BBa_K1178000

tRNA and synthetase for 3,4-dihydroxy-L-phenylalanine (L-DOPA) incorporation at UAG codon

L-DOPA

The part includes proper promoters and terminator along with the gene coding for the Methanocaldococcus jannaschii synthetase mutated to charge the orthogonal tRNA (also in the part) with the non-canonical amino acid 3,4-dihydroxy-L-phenylalanine (L-DOPA). This part encodes for the incorporation L-DOPA during translation at AUG Amber stop codons. For more information on non-canonical amino acid incorporation click [http://2013.igem.org/Team:Greensboro-Austin/ncAAs here].

These data show that our tRNA/synthetase pair is suppressing the Amber (UAG) stop codon, but they do not definitively show that it is L-DOPA that is being incorporated. It is possible that the synthetase is leaky and incorporates tyrosine or other substrates at these codons. In fact data from the original paper and our independent observation support the conclusion that this synthetase charges both tyrosine and L-DOPA to its tRNA. An improved synthetase would be valuable for the production of proteins which specifically require L-DOPA for function. This part will serve as an important starting point for teams wishing to make the few mutations required to incorporate a wide variety of ncAAs into proteins, most of which work with much higher fidelity than the L-DOPA synthetase.

1. Alfonta et al. Site-specific incorporation of a redox-active amino acid into proteins. J Am Chem Soc. 2003 Dec 3;125(48):14662-3. PubMed PMID: 14640614.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1154
  Illegal BamHI site found at 1160
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1186
  Illegal NgoMIV site found at 1646
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 317
  Illegal SapI.rc site found at 1110