JoVE Encyclopedia of Experiments
Microbiology
0 views • 3:09 min • November 28th, 2025
Take a genetically engineered E. coli culture expressing a chromosomal tetracycline operator (tetO) array.
The bacteria also carry a plasmid with an arabinose-inducible promoter that drives the expression of YFP-tagged tetracycline repressor protein.
Transfer the culture into an enriched nutrient medium containing antibiotics, and incubate with shaking to select plasmid-bearing cells.
As the cells grow, replisomes form and initiate chromosomal DNA replication.
At the exponential phase, collect a portion as the uninduced control.
Add arabinose, an inducer, to the remaining culture and incubate both cultures with shaking.
In the uninduced culture, the AraC dimer remains bound to the DNA loop, inhibiting repressor expression.
In the induced culture, arabinose binds to the AraC dimer, inducing repressor expression.
The repressor proteins bind to the chromosomal tetO array, halting replication fork progression.
Use fluorescence microscopy to observe both cultures.
Fluorescent foci in the induced E. coli culture confirm repressor protein-mediated replication blockage.
An E. coli strain carrying a tetracycline operator array in the pKM1 plasmid is used for this procedure. pKM1 encodes for TetR-YFP,
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