This study investigates the role of a tetracycline repressor protein in E. coli by utilizing fluorescence microscopy to observe its effects on chromosomal DNA replication. The experiment involves genetically engineered E. coli cultures that express a chromosomal tetracycline operator array and an arabinose-inducible promoter.
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, an inducible yellow fluorescent protein-tagged tetracycline repressor protein.
Dilute a fresh overnight culture of this E. coli strain to an optical density at 600 nanometers of 0.01 in a dilute complex medium with antibiotics as required for selection. Grow the culture at 30 degrees celsius with shaking to an optical density at 600 nanometers of 0.05 to 0.1. Remove a 10 milliliter sample to serve as the uninduced control.
Add 0.01% arabinose to the remaining culture to induce the production of TetR-YFP from pKM1. Continue to grow both the uninduced and induced cultures at 30 degrees celsius with shaking. After one hour, check for the presence of a single focal point within each cell of the induced culture using fluorescence microscopy.
If the induced cells are confirmed blocked, indicated by more than 70% of cells having a single focus, record the optical density and remove 7.5 milliliters of the sample for analysis by two-dimensional gel electrophoresis. Remove an equivalent sample from the uninduced control culture.
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