In order to have the characteristics above a

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In order to have the characteristics above, a tRNA/synthetase pair of M. jannaschii was imported into E. coli . This was totally legal because (1) cross-species aminoacylation was inefficient and (2) the anticodon loop was not a key determinant of synthetase recognition. M. jannaschii ’s tRNA Tyr E. coli tRNA Tyr , its first base pair of the acceptor stem was different (CG instead of GC in E. coli ), and its synthetase (TyrRS) had a minimalist anticodon loop binding domain. Also - (3) there was no editing mechanism / proofreading to correct the addition of O-methyl-
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tyrosine. Library Generation (tRNA) : to further reduce recognition of M. jannaschii tRNA by E. coli synthetases, 11 nucleotides of the tRNA that didn’t interact with TyrRS were randomly mutated. A library was generated and passed through a series of selections. Negative selection , suppression of amber in the barnase gene The amber nonsense codon is chilling in the middle of the gene coding for barnase, which is TOXIC. We’re trying to select for tRNA that isn’t aminoacylated by E. coli synthetases. The ones that are aminoacylated will add an amino acid where the stop codon is, effectively transcribing the barnase gene and causing cell death. Effect: Colonies grew if they didn’t transcribe barnase. This was only possible if they contained tRNAs that avoided aminoacylation by E. coli synthetases. We got rid of the tRNAs that were. Positive selection , suppression of amber in the β-lactamase gene Now, the goal is not to remove tRNA recognized by E. coli synthetase, but rather identify the ones that are efficiently aminoacylated by TyrRS. You want to see tRNAs insert an amino acid (instead of terminating the gene) so that β-lactamase is expressed and cells can live on a medium containing AMPICILLIN. Efficiency was also tested using an in vivo complementation assay with a pBLAM plasmid. Library Generation (TyrRS) : Using a crystal structure of a homologous TyrRS from Bacillus stearothermophilus , five residues in the active site were mutated to alanine. This model was used as a template for PCR random mutagenesis with doped oligonucleotides. Positive selection , suppression of amber at NONESSENTIAL position in CAT gene We’re now testing the mutant TyrRS for its ability to charge the orthogonal mutRNA. If it charges any amino acid, the cell produces CAT and survives.
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The surviving cells were grown in the ABSENCE of O-methyltyrosine and chloramphenicol. The cells that did not survive were isolated; this means that TyrRS was evolved to deliver O-methyltyrosine, specifically, to the amber stop codon. Mutant TyrRS genes were taken from the cells, recombined in vitro, and transformed into E. coli for multiple rounds of selection. The concentration of chloramphenicol was gradually increased until a clone whose survival was dependent on O-methyltyrosine was isolated.
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