Methods and Compositions for the Production of Orthogonal tRNA-Aminoacyl tRNA Synthetase Pairs

Abstract

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

1 .- 114 . (canceled) 
     
     
         102 . A method for identifying an orthogonal tRNA-tRNA synthetase pair for use in an in vivo translation system of a second organism, the method comprising:
 introducing a marker gene, a tRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from a first organism into a first set of cells from the second organism;   introducing the marker gene and the tRNA into a duplicate cell set from the second organism; and   selecting or screening for surviving cells or for cells showing a specific screening response in the first set that fail to survive or show said response in the duplicate cell set, wherein the first set and the duplicate cell set are grown in the presence of a selection or screening agent, wherein the surviving or screened cells comprise the orthogonal tRNA-tRNA synthetase pair for use in the in the in vivo translation system of the second organism.   
     
     
         103 . The method of  claim 102 , wherein the comparing and selecting or screening comprises an in vivo complementation assay. 
     
     
         104 . The method of  claim 102 , wherein concentration of the selection or screening agent is varied. 
     
     
         105 . The method of  claim 102 , wherein the first organism is a prokaryotic organism. 
     
     
         106 . The method of  claim 102 , wherein the second organism is a prokaryotic organism. 
     
     
         107 . The method of  claim 102 , wherein the first and second organism are different. 
     
     
         108 . The method of  claim 102 , wherein the first organism is selected from the group consisting of:  Methanococcus jannaschii, Methanobacterium thermoautotrophicum , and a  Halobacterium.    
     
     
         109 . The method of  claim 102 , wherein the second organism is  Escherichia coli.    
     
     
         110 . The method of  claim 102 , wherein the selecting or screening comprises one or more positive or negative selection or screening chosen from the groups consisting of: a change in amino acid permeability, a change in translation efficiency, and a change in translational fidelity, and wherein the one or more change is based upon a mutation in one or more gene in an organism in which an orthogonal tRNA-tRNA synthetase pair are used to produce protein. 
     
     
         111 . The method of  claim 102 , wherein the selecting or screening comprises selecting or screening at least 2 selector codons within one or more selection gene or within one or more screening gene. 
     
     
         112 . The method of  claim 111 , wherein the at least 2 selector codons are in the same selection gene or the same screening gene. 
     
     
         113 . The method of  claim 111 , wherein the at least 2 selector codons are in different selection or screening genes. 
     
     
         114 . The method of  claim 111 , wherein the at least 2 selector codons comprise different selector codons. 
     
     
         115 . The method of  claim 111 , wherein the at least 2 selector codons comprise the same selector codons.