Part:BBa_K1492002
Methanococcus jannaschii tyrosyl-tRNA synthetase
tRNA synthetase for p-Azido-L-Phenylalanine
Usage and Biology
iGEM team TU Eindhoven 2014 uses Methanococcus jannaschii tyrosyl-tRNA synthetase(Mj TyrRS) for the introduction of a phenylalanine like amino acid into a membrane protein. The non-natural amino acid that has been used is p-Azido-L-phenylalanine (pAzF).
The complete tRNA system/plasmid has been given to us by Peter G. Schultz et al.(1)
This system contains pylT tRNA and two times TyrRS.
(1) Jason W. Chin, Stephen W. Santoro, Andrew B. Martin, David S. King, Lei Wang, and Peter G. Schultz, “Addition of p-Azido-L-phenylalanine to the Genetic Code of Escherichia coli”, journal of the American Chemical Society. 124 (2002), 9026-9027
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 52
Illegal SapI.rc site found at 84
Illegal SapI.rc site found at 877
Characterization:
To prove whether or not the tRNA synthase works, an assay has been done in which the pAzF has been used. The function is characterized by the expression of the protein with the non-natural amino acid, because it was not possible to examine the expression of the tRNA synthetase itself. As long as the protein is expressed, the tRNA synthetase must work else the amino acid will not be incorporated and the transcription will terminate at the TAG codon. The non-natural amino acid that has been used, p-Azido-L-phenylalanine (pAzF), only reacts with a DBCO group. For this assay fluorescently labeled DBCO has been used to characterize the presence of pAzF. For the protocol used, see: http://2014.igem.org/Team:TU_Eindhoven/Protocols -> FACS for sorting with DBCO-TAMRA.
Figure 1: COMPx labeled with DBCO-PEG4-TAMRA
Figure 2: COMPy labeled with DBCO-PEG4-TAMRA
The results show that in two types of proteins the non-natural amino acid is present. (Figure 1 and 2) Therefore the tRNA synthetase must work properly.
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