A general approach for the generation of orthogonal tRNAs

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A general approach for the generation of orthogonal tRNAs Lei Wang, Peter G Schultz Chemistry & Biology Volume 8, Issue 9, Pages 883-890 (September 2001) DOI: 10.1016/S1074-5521(01)00063-1

Fig. 1 Anticodon-loop tRNA library (left) and all-loop tRNA library (right) derived from M. jannaschii tRNATyrCUA. Randomly mutated nucleotides (N) are shaded in black. Chemistry & Biology 2001 8, 883-890DOI: (10.1016/S1074-5521(01)00063-1)

Fig. 2 A general selection for suppressor tRNAs that are poor substrates for the E. coli synthetases and charged efficiently by a cognate synthetase of interest. Chemistry & Biology 2001 8, 883-890DOI: (10.1016/S1074-5521(01)00063-1)

Fig. 3 Negative selection based on the suppression of two amber codons in the barnase gene. Selections were carried out in GMML liquid medium, and 20 mM of arabinose was used to induce barnase expression. Chemistry & Biology 2001 8, 883-890DOI: (10.1016/S1074-5521(01)00063-1)

Fig. 4 Positive selection based on the suppression of an amber codon in the β-lactamase gene. pAC plasmid encoding the suppressor tRNA was cotransformed with pBLAM-JYRS in E. coli DH10B cells: (A) selection was carried out in liquid 2×YT media with various concentrations of ampicillin; (B) selection was carried out on 2×YT agar plates with 500 μg/ml ampicillin. Chemistry & Biology 2001 8, 883-890DOI: (10.1016/S1074-5521(01)00063-1)

Fig. 5 DNA sequences of mutant suppressor tRNAs selected from anticodon-loop library and all-loop library. JY stands for the wild-type M. jannaschii tRNATyrCUA. Wild-type nucleotides are shaded in blue; mutated nucleotides in red; and semi-conserved nucleotides in gray. Chemistry & Biology 2001 8, 883-890DOI: (10.1016/S1074-5521(01)00063-1)