This study investigates the modulation of intracellular cyclic di-GMP levels in genetically modified pathogenic bacteria using fluorescence measurements. The approach utilizes a GFP reporter system to assess the impact of various test compounds on bacterial behavior.
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 a wavelength of 600 nanometers with a setting of 10 flashes per well and a settling time of 0.2 seconds. Prior to measuring fluorescence, make an automatic gain and focal adjustment based on the negative control well A24 and set the gain target value at 75%.
Select focus adjustment and a channel A on the right of the window. Measure the fluorescence from the GFP reporter at an excitation maximum of 485 nanometers and an emission maximum of 520 nanometers, with a setting of 10 flashes per well and a settling time of 0.2 seconds.
Collect data from all plates examined. Data readings are automatically saved as default by the plate reader control software.
To analyze the data, view the readings by clicking on the Mars icon within the control software to open the statistical analysis package. Retrieve data by double-clicking on the plate number of interest to access the data for analysis.
Evaluate the uniformity and reproducibility of the assay using a robust Z prime analysis.
Then, calculate percent inhibition of growth and assess the percent inhibition of the intracellular level of c-di-GMP as detailed in the text protocol.
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