Tightly controlled two-stage expression vectors employing the Flp/FRT-mediated inversion of cloned genes.

We have developed a tightly controlled, two-stage expression system. It is based on a single plasmid that carries the TetR repressor/Ptet promoter/Otet operator for the first-stage control, and the Flp recombinase/ FRT sites for the second-stage control. The gene to be expressed (GENE) is cloned in an inverted orientation (with respect to the stationary promoter) into a multiple-cloning site (MCS) located between two convergent FRT1 and FRT2 sites. In the OFF stage, no inadvertent transcription can enter the 5' end of cloned GENE because of four rrnBT1 terminators, located just outside the FRT1-MCS-FRT2 cassette and because the FRT2 construct was deprived of any promoter function. When using the lacZ reporter, it was shown that in their OFF stage our two-stage expression plasmids exhibit a significantly lower basal expression than the repressed single-stage tetR/PtetOtet-lacZ vectors. To enter the ON stage, the tetR/PtetOtet module is induced by adding autoclaved chlortetracycline (cTc), leading to synthesis of the Flp recombinase, which in turn, inverts the FRT1-MCS-FRT2 module together with the cloned GENE. This results in the massive GENE expression from one (pInvMS) or two (pImpMS) stationary promoters.