Home › Microbiology › Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa
Microbiology JoVE (Open Access) Citable · DOI

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

DOI: 10.3791/54115-v
What you'll learn
  • ✓ Establish a GFP-tagged c-di-GMP reporter strain of Pseudomonas aeruginosa
  • ✓ Execute high-throughput screening of small molecule libraries in 384-well plates
  • ✓ Measure intracellular c-di-GMP levels and identify bioactive compounds
  • ✓ Analyze growth and signaling data to discover c-di-GMP modulators
Protocol

Biopharma Insights This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Difficulty
advanced
Total time
~3–4 days (strain construction + overnight culture + plate incubation + analysis)
Model organism
Pseudomonas aeruginosa (bacterial strain, GFP-tagged)
Biosafety
BSL-2

Steps

1
Generate GFP-tagged c-di-GMP reporter strain

Construct a Pseudomonas aeruginosa strain with GFP reporter linked to c-di-GMP signaling. This engineered strain enables fluorescence-based detection of intracellular cyclic di-GMP levels.

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2
Prepare starter culture for inoculation

Grow an overnight culture of the reporter P. aeruginosa strain to achieve target cell density. Dilute or adjust culture as needed for consistent inoculation into screening plates.

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3
Inoculate 384-well plates with small molecules

Distribute starter culture into 384-well plates containing arrayed small molecule compounds at defined concentrations. Incubate plates under controlled conditions to allow bacterial growth and compound exposure.

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4
Measure growth and intracellular c-di-GMP levels

Quantify optical density (growth) and GFP fluorescence intensity (c-di-GMP levels) across all wells using a plate reader. Normalize fluorescence to cell density to determine compound effects on signaling.

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5
Identify hit compounds from screening results

Apply statistical and computational analysis to raw screening data to identify small molecules that significantly modulate c-di-GMP levels. Rank candidates by potency and selectivity for downstream validation.

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