Part:BBa_K1223014

tRNA-Pyl (pylT) gene from Methanosarcina barkeri str. Fusaro

this is the DNA coding sequence for the tRNA of pyrrolysine from the Archaea Methanosarcina barkeri str. Fusaro.

this tRNA is a part of the machinery that is used to incorporate pyrolysine into proteins in E.coli.

the tRNA synthetase that charges the tRNA with pyrrolysine can be found in biobrick BBa_k1223013.

the tRNA has the anticodon CUA - which means that the sense codon needed to incorporate pyrrolysine is TAG - the amber stop codon.

Design Notes

this is a part of the pyrrolysine incorporation machinery.

this tRNA is charged by pyrrolysyl-tRNA synthetase (pylS gene) to incorporate the amino acid pyro lysine. the tRNA has CUA anticodon that recognizes the TAG (UAG) codon on the mRNA during the translation process.

pylS CDS can be found in part BBa_K1223013.

Applications of BBa_K1223014

This part was used by us to incorporate the unnatural amino acid propargyl-L-lysine into various proteins. in the fluorescent gel we show incorporation of the unnatural amino acid into copper oxidase of E.coli in various loactions

H117,N262,D411,M412 - position and amino acid that was replaced with the UAA. CueO - native protein without UAA incorporated. L - ladder.

we used a reactive fluorescent dye to identify the incorporated UAA in the gel.

Source

derived from the genomic sequence of Methanosarcina barkeri. gene name - pylT

clasification: Archaea : Euryarchaeota : Methanomicrobia : Methanosarcinales : Methanosarcinaceae : Methanosarcina

References

Srinivasan,G., James,C.M. and Krzycki,J.A., Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA, Science 296 (5572), 1459-1462 (2002) Milles, S.; Tyagi, S.; Banterle, N.; Koehler, C.; VanDelinder, V.; Plass, T.; Neal, A. P.; Lemke, E. A., Journal of the American Chemical Society 2012, 134 (11), 5187-95.

Usage and Biology

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]