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

Abstract

Source: Rugjee, K. N., et al. Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa. J. Vis. Exp. (2016).

This video demonstrates the screening of cyclic di-GMP modulators using genetically modified bacteria that express green fluorescent protein (GFP) under the control of a c-di-GMP-sensitive promoter. Fluorescence is measured in a multi-well plate containing different test compounds. Compounds that inhibit c-di-GMP production lead to reduced fluorescence. The data is then analyzed to identify potential modulators of c-di-GMP signaling in bacteria.

Protocol

1. Generation of Green Fluorescent Protein (GFP) Tagged cyclic di-GMP (c-di-GMP) Reporter Pseudomonas aeruginosa Strain

1. Inoculate 5 ml of lysogeny broth (LB) medium with a single colony of P. aeruginosa and incubate overnight at 37 °C with shaking at 200 rpm.
2. Transfer 2 ml of the overnight culture into 200 ml of fresh LB medium in a 1 L flask and incubate at 37 °C with shaking at 200 rpm.
3. Monitor optical density at 600 nm (OD₆₀₀) every 30 min by taking a 1 ml sample from the flask and examining it using a spectrophotometer.
4. When the OD₆₀₀ reaches between 0.3 ....

Results

Figure 1. A 384-well Plate Map Used for the Small Molecule Screening Assay. The compounds from t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lysogeny Broth (Lennox)	Sigma Aldrich	L3022-1KG
Sucrose	Sigma Aldrich	S0389-1KG
Lysogeny Broth with Agar (Lennox)	Sigma Aldrich	L2897-1KG
Ampicillin sodium	Sigma Aldrich	A0166-25G
Glycerol	Sigma Aldrich	G5516-1L
Phosphate Buffered Saline, pH 7.4	Life technologies	10010-056
Tobramycin sulfate	Sigma Aldrich	T1783-100MG
Dimethyl Sulfoxide (DMSO)	Sigma Aldrich	276855-250ML
Genesys 10S UV-Vis spectrophotometer	Thermoscientific	840-208100
2 mm gap electroporator cuvette	Bio-Rad	1652092
BioRad GenePulser XCell electroporator	Bio-Rad	1652662
Leica Fluorescent Stereomicroscope	Leica Microsystems	MZ16FA
BRAND magnetic stirring bar, PTFE, cylindrical with pivot ring (sterilize by autoclaving before use)	Sigma Aldrich	Z328952-10EA
384-well, white-walled, clear-bottom plates	Greiner	781098
Multidrop Combi reagent dispenser	Thermo Scientific	5840300
Multidrop Combi tubing	Thermo Scientific	24073290
VIAFLO II 16-channel electronic pipette	Integra Biosciences	4642
100 ml sterile disposable reagent reservoirs	Fisher Scientific	12399175
AeraSeal air-permeable membranes	Sigma Aldrich	MKBQ1886
Pherastar plate reader (Software version: 4.00 R4, Firmware version: 1.13)	BMG Labtech	Pherastar FS