Team:TU Eindhoven/Achievements/Submitted Parts/tRNA

Mj TyrRS - BBa k1492002

This year’s iGEM team of TU Eindhoven 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. This system contains pylT tRNA and two times Mj TyrRS.

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. Check our Protocol Page for all the protocols of TU Eindhoven 2014.

The results show that in two types of proteins the non-natural amino acid is present (Figure 1 and Figure 2). Therefore the tRNA synthetase must work properly.

tRNA

The tRNA (pylT) used was already in the parts iGEM data bank (see BioBrick) Results in Figure 1 and Figure 2 show that the tRNA works with the non-natural amino acid, pAzF, used by team TU Eindhoven 2014.

Bibliography

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.