Abstract: To obtain amber suppressor tRNA with high orthogonality,a novel inducible high-copy-number screening plasmid was constructed.The oriL were placed under the control of Lac promoter so that IPTG could induce the plasmid replication with a high copy number.When the plasmid contained the amber suppressor tRNA with poor orthogonality,a large numberss.When the plasmid contained the amber suppressor tRNA with poor orthogonality,a large number of tRNA transcrips led to host cell dedth.Only the host containing the suppressor tRNA with high orthogonality could survive.With the screening system,the mutant liberary of amber suppressor tRNA was screened,and amber suppressor tRNA mutant with high orthogonality was obtained.