Screening of Cyclic di-GMP Modulators Using Bacteria Expressing a Fluorescent Reporter Protein

Take a multi-well plate containing genetically modified pathogenic bacteria grown with different test compounds, along with negative controls.

The bacteria carry a plasmid with a gene encoding the green fluorescent protein or GFP.

The promoter for the gene is activated by a protein that responds to intracellular cyclic di-GMP or c-di-GMP concentrations.

Place the plate in a plate reader.

Use the negative control, which fluoresces maximally due to the absence of a c-di-GMP inhibitor, to adjust the settings for optimal fluorescence signal measurement.

If a test compound inhibits c-di-GMP formation, the intracellular c-di-GMP level decreases.

Reduced c-di-GMP levels trigger a conformational change in the protein, leading to repression of the promoter and low GFP expression, which results in decreased fluorescence.

Measure the fluorescence and analyze the data to identify test compounds that modulate intracellular c-di-GMP levels in pathogenic bacteria.

Approximately 30 minutes before spectrophotometric measurement, pre-warm the plate reader to 37 degrees Celsius to avoid condensation. Gently remove the air-permeable cover seal from the plate before reading.

Then, measure the optical density at