Copy-Control Tightly Regulated Expression Vectors Based on pBAC/oriV

Abstract

A novel type of expression vectors with a dual regulation of both the plasmid copy number and gene expression, is described. The most important and beneficial feature of these vectors is that when they are not induced, they are maintained as a single-copy plasmid, and therefore, any residual expression is much more tightly regulated than for the conventional multicopy expression vectors. The simplest version of these copy-control expression vectors is based on the pBAC/oriV plasmid that carries the trfA up-mutant gene under control of the L-arabinose-inducible Para promoter (araC-P _BAD ). The same promoter controls expression of a gene cloned into MCS. Thus, addition of the inducer (L-arabinose) simultaneously turns on amplification of the plasmid and expression of the cloned gene. Net result is about a 50,000-fold increase in the cloned gene expression. However, when not induced, background expression level is very low, which is important for the maintenance of any “toxic” genes. This vector could be used in most E. coli hosts. Similar versions of the described vector employ the rhamnose-inducible P _rha promoter (rhaS-P _rha ). Other expression systems allow independent regulation of the plasmid amplification and of the cloned gene expression, and some also use the P _LtetO-1 promoter. Copy-control expression vector pETcoco, based on the pT7lacO promoter, is commercially available.