Disruption of Chlamydia trachomatis Essential Genes Using DOPE (Dependence on Plasmid-Mediated Expression).

We describe in detail an experimental strategy, DOPE (Dependence On Plasmid-mediated Expression), to enable disruption of an essential chlamydial gene. A wild-type Chlamydia trachomatis strain is first transformed with a β-lactamase-selectable plasmid carrying a copy of the gene under the inducible control of an anhydrotetracycline (ATC)-responsive promoter. Transformants are selected under permissive conditions in the absence of ATC to establish stable plasmid maintenance. The resulting chlamydiae are subsequently transformed with a suicide plasmid encoding a group II intron engineered to target the chromosomal copy of the gene. Secondary transformants with the intron inserted into the chromosomal locus are then selected with spectinomycin in the presence of ATC to induce expression of the plasmid-encoded allele. This protocol thus produces a conditional knockout strain in which expression of the essential gene is inducibly controlled by ATC so that its function in chlamydial growth and developmental progression can be delineated.