可诱导高拷贝的正交琥珀抑制性tRNA筛选质粒的构建

Screening system for amber suppressor tRNA based on inducible high-copy-number plasmid

为筛选获得对大肠杆菌氨酰合成酶具有高度正交性的琥珀抑制tRNA,建立了一种基于可诱导高拷贝质粒的筛选系统。利用乳糖操纵子对oriL复制子进行调控,有IPTG存在时使其携带的琥珀抑制性tRNA大量转录。正交性不高的琥珀抑制性tRNA大量转录时导致宿主菌死亡,只有携带高正交性的琥珀抑制性tRNA的克隆可以存活,从而达到筛选目的。利用这一筛选质粒,构建了琥珀抑制性tRNA突变文库,并进行了筛选,获得了与大肠杆菌氨酰tRNA合成酶具有高度正交性的突变体,并利用含琥珀抑制突变的半乳糖苷酶突变体进行了验证,证实利用可诱导高拷贝的筛选质粒可筛选出高正交性的琥珀抑制性tRNA突变体。

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.