PBR322 vectors having tetracycline-dependent replication

Abstract

Few Escherichia coli cloning vectors are available that can both be stably maintained and efficiently cured. One such vector is pAM34, a pBR332 derivative constructed by Gil and Bouché (1991). Replication of this plasmid is driven by the lacZYA promoter under control of a gratuitous inducer. However, lac operator–repressor interactions are also used to regulate many expression systems which limit the utility of pAM34. In this report pAM34 has been modified by replacement of the lac regulatory elements with those of the transposon Tn10 tetracycline resistance module. This resulted in medium copy number plasmids that are dependent on the presence of tetracycline (or less satisfactorily, anhydrotetracycline) for replication. The tetracycline-dependent plasmids are rapidly lost in the absence of tetracycline and plasmid loss is markedly accelerated when the host strain expresses a tetracycline efflux pump.