Incorporation of unnatural nucleotides and methods thereof

Abstract

Disclosed herein are methods, compositions and kits for the synthesis of proteins which comprises unnatural amino acids that utilize a mutant tRNA.

1 .- 23 . (canceled) 
     
     
         24 . An  E. coli  cell comprising:
 (a) a nucleoside triphosphate transporter from  Phaeodactylum tricornutum;      (b) an mRNA comprising an unnatural base comprising a structure selected from   
       
         
           
           
               
               
           
         
         (c) a tRNA comprising an unnatural base comprising a structure selected from 
       
       
         
           
           
               
               
           
         
       
       and
 (d) an aminoacyl tRNA synthetase derived from an organism selected from the group consisting of  Methanosarcina barkeri  and  Methanococcus jannaschii.    
 
     
     
         25 . The  E. coli  cell of  claim 24 , wherein the cell further comprises an oligonucleotide encoding the mRNA. 
     
     
         26 . The  E. coli  cell of  claim 24 , wherein the cell further comprises an oligonucleotide encoding the tRNA. 
     
     
         27 . The  E. coli  cell of  claim 24 , wherein the cell further comprises an oligonucleotide encoding the aminoacyl tRNA synthetase. 
     
     
         28 . The  E. coli  cell of  claim 24 , wherein the cell further comprises a single oligonucleotide encoding the mRNA and the tRNA. 
     
     
         29 . The  E. coli  cell of  claim 24 , wherein the cell further comprises a single oligonucleotide encoding the mRNA, the tRNA, and the aminoacyl tRNA synthetase. 
     
     
         30 . The  E. coli  cell of  claim 24 , wherein the mRNA comprises one or more codons selected from AXC and GXC, wherein X is the unnatural base. 
     
     
         31 . The  E. coli  cell of  claim 30 , wherein the mRNA comprises the codon AXC. 
     
     
         32 . The  E. coli  cell of  claim 31 , wherein the unnatural base comprises the structure 
       
         
           
           
               
               
           
         
       
     
     
         33 . The  E. coli  cell of  claim 31 , wherein the unnatural base comprises the structure 
       
         
           
           
               
               
           
         
       
     
     
         34 . The  E. coli  cell of  claim 30 , wherein the mRNA comprises the codon GXC. 
     
     
         35 . The  E. coli  cell of  claim 34 , wherein the unnatural base comprises the structure 
       
         
           
           
               
               
           
         
       
     
     
         36 . The  E. coli  cell of  claim 34 , wherein the unnatural base comprises the structure 
       
         
           
           
               
               
           
         
       
     
     
         37 . The  E. coli  cell of  claim 24 , wherein the aminoacyl tRNA synthetase is derived from  Methanosarcina barkeri.    
     
     
         38 . The  E. coli  cell of  claim 36 , wherein the mRNA comprises an unnatural base comprising the structure 
       
         
           
           
               
               
           
         
       
       and the tRNA comprises an unnatural base comprising the structure 
       
         
           
           
               
               
           
         
       
     
     
         39 . The  E. coli  cell of  claim 37 , wherein the mRNA comprises one or more codons selected from AXC and GXC, wherein X is the unnatural base. 
     
     
         40 . The  E. coli  cell of  claim 24 , wherein the aminoacyl tRNA synthetase is derived from  Methanococcus jannaschii.    
     
     
         41 . The  E. coli  cell of  claim 39 , wherein the mRNA comprises an unnatural base comprising the structure 
       
         
           
           
               
               
           
         
       
       and the tRNA comprises an unnatural base comprising the structure 
       
         
           
           
               
               
           
         
       
     
     
         42 . The  E. coli  cell of  claim 40 , wherein the mRNA comprises one or more codons selected from AXC and GXC, wherein X is the unnatural base. 
     
     
         43 . The  E. coli  cell of  claim 24 , wherein the tRNA is derived from an organism selected from the group consisting of  Methanosarcina barkeri  and  Methanococcus jannaschii.