Tightly controlled two-stage expression vectors employing the Flp/FRT-mediated inversion of cloned genes
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We have developed a tightly controlled, two-stage expression system. It is based on a single plasmid that carries the TetR repressor/P tet promoter/O tet 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 convergentFRT1 andFRT2 sites. In the OFF stage, no inadvertent transcription can enter the 5′ end of clonedGENE because of fourrrnBT1 terminators, located just outside theFRT1-MCS-FRT2 cassette and because theFRT2 construct was deprived of any promoter function. When using thelacZ reporter, it was shown that in their OFF stage our two-stage expression plasmids exhibit a significantly lower basal expression than the repressed single-stagetetR/P tetOtet-lacZ vectors. To enter the ON stage, thetetR/P tetOtet module is induced by adding autoclaved chlortetracycline (cTc), leading to synthesis of the Flp recombinase, which in turn, inverts theFRT1-MCS-FRT2 module together with the clonedGENE. This results in the massiveGENE expression from one (pInvMS) or two (pImpMS) stationary promoters.
Index EntriesEscherichia coli two-stage control tet promoter inversion cassette
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