Expression plasmid with a very tight two-step control: Int/att-mediated gene inversion with respect to the stationary...

A very tightly controlled expression vector was constructed, which was originally designed as to be able to use any promoter, constitutive or regulated. Moreover, in vector pNH46T1, the repressible P(tac)/P(lac) promoters were used to transcribe genes cloned in the proximal multiple cloning site (MCS), which was flanked by convergent attB and attP sites. The gene of interest was cloned into MCS in the OFF orientation, i.e. facing the promoter(s). In such OFF orientation, the cloned gene could not be expressed, and only its anti-sense mRNA could be produced. Four strong rrnBT1 terminators, in a tandem arrangement and proximal to the N-terminal end of the cloned non-inverted gene, were protecting it from any inadvertent transcription originating in the vector. Moreover, the P(tac)/P(lac) promoters/operators are controlled by the LacI(q)ts and LacI(+) repressor(s) that further reduce the basal gene expression in the uninduced state. When induced, the total vector population is converted to the ON orientation by expression of the Int function that inverts the attB and attP-flanked MCS including the cloned gene. This places the gene under direct control of the P(tac)/P(lac) promoters, and thus results in very high expression. An additional feature is the anti-termination system that consists of the promoter-proximal nutL site and the inducible gene N, whose role in the ON state is to overcome the rrnBT1 terminators and any other adventitiously cloned terminators.