Part:BBa_K1223013

Pyrolysyl-tRNA synthetase CDS

this part is the coding sequence for the Pyrolysyl-tRNA synthetase enzyme from the archaea Methanosarcina barkeri str. fusaro. The enzyme 'load' the amino acid pyrolysine onto its dedicated tRNA for subsequent incorporation into proteins during the translation process. The tRNA synthetase is part of the unnatural amino acid (UAA) incorporation machinery along with the dedicated tRNA molecule (BBa_K1223014).

Design Notes

CDS of the pyrolysyl-tRNA synthetase (pylS) that charges the pyrolysine tRNA (pylT) with pyrrolysine.

Applications of BBa_K1223013

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 - pylS

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 394
  Illegal BglII site found at 755
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 90
  Illegal BsaI.rc site found at 128