Tyrosyl- and Pyrrolysyl-tRNA Synthetases
Figure 1: Tyrosine-binding site in apo M. jannaschii TyrRS
The electrostatic distribution around the tyrosine-binding site is shown. Positive potential is blue (10 mV), neutral potential (0 mV) is white, and negative potential (−10 mV) is red (Zhang et al.,2005).
Figure 2: Pyrrolisine-binding site of wild type Methanosarcina mazei PylRS. Structural composition of the active site of PylRS with activated Pyl (Pyl-AMP) as a substrate (Guo et al., 2014).
Generating the Library
Modification of the aaRS
Figure 3: Adaption of an orthogonal aminoacyl-synthetase through positive and negative selection cycles.
The positive-selection plasmid (BBa_K2201900 in pSB3T5)contains a kanamycin resistance with amber stop codons. After cotransformation with the library plasmid only cells which contain a synthetase which incorporates any amino acid in response to the amber codon survive. All surviving clones contain an aaRS that incorporates the target ncAA or any endogenous amino acid. The negative selection plasmid (BBa_K2201901 in pSB3T5) is used for the selection for the specificity of the clones. The target ncAA is not supplemented in the media. When incorporating endogeneous amino acids, the barnase is expressed through an amber codon and the cell dies.
Modification of the tRNA
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