Quorum LuxO: Assay for change of GFP production in the absence of autoinducers using SL353 (luxOD47E, qrr4-gp) Measured in Microorganism System Using Plate Reader - 2132-06_Inhibitor_Dose_DryPowder_Activity
Assay Overview: This is an assay that will help to define the mechanism of action for a probe candidate. We will test compounds that presumably act on or downstream of LuxO in a V. cholerae luxOD47E mutant carrying a qrr4-gfp transcriptional fusion green fluorescent protein (GFP) reporter (strain SLS353). In this strain, the luxOD47E mutant mimics the phosphorylated active form of LuxO and is more ..
BioActive Compounds: 3
Depositor Specified Assays
Keywords:Vibrio cholerae, quorum sensing
Assay Overview: This is an assay that will help to define the mechanism of action for a probe candidate. We will test compounds that presumably act on or downstream of LuxO in a V. cholerae luxOD47E mutant carrying a qrr4-gfp transcriptional fusion green fluorescent protein (GFP) reporter (strain SLS353). In this strain, the luxOD47E mutant mimics the phosphorylated active form of LuxO and is constitutively active. In addition, expression of GFP is controlled by the qrr4 promoter, which is positively regulated by LuxO~P. Thus, if a given compound decreases GFP production in this stain, it is inferred that it acts on LuxO or downstream of LuxO (i.e. acting on qrr RNAs or Hfq).
Expected Outcome: The strain has a basal level of GFP expression. Compounds that act as a LuxO inhibitor will cause a decrease in GFP levels leading to a decrease in fluorescence. A compound causing no change in GFP could act elsewhere but still will be of interest as a probe candidate.
Vibrio cholerae quorum-sensing modulator bioassay
This protocol is enough for 50 384-well plates. It can be scaled up or down accordingly.
1. For luxO, qrr and Hfq inhibitors: SLS353 (V. cholerae luxOD47E carrying a qrr4:GFP reporter construct)
LB Medium: Dissolve in 10 g/L Tryptone, 5 g/L Yeast Extract, and 10 g/L NaCl in distilled water, autoclave, store at room temperature
Kanamycin (10 mg/mL): Dissolve 10 mg tetracycline in 1 mL water, store at -20 oC.
LB/Kan: . Final concentration of Kanamycin is 100 microg/mL. Use it fresh
CAI-1 stock: Dissolve CAI-1 in DMSO to 50 mM (10.7 mg/mL), store at -20oC
1. Grow up each reporter strain in 50 mL LB/Kan at 30oC for >16 hours with shaking (200 rpm). The final OD600 of each culture should be > 3.0
2. Add 40 mL of the culture to 1000 mL of LB/Kan, mix well. (Note: avoid biofilm aggregates in the culture, a low speed centrifugation (200 rpm for 1 min) should remove most aggregates)
3. Dispense 50 microL of diluted culture into each well of a 384 well plate (Black with clear bottom. We use Greiner 781096 plates for previous assays. We have screened compounds at 20 microM final concentration. Add CAI-1 as positive control for BH1578 (need to adjust concentration))
4. Incubate the plates at 30oC without shaking for 8-16 hours. (I usually stack up 10 plates and cover the top plate only)
5. Measure GFP fluorescence and OD600 in a Perkin-Elmer Envison Multilabel Plate Reader
The assay is looking for a decrease in green fluorescent
protein (GFP) signal in a bacterial strain of Vibrio cholerae. Any actives are likely to be inhibiting a kinase, luxO or an RNA chaperone, Hfq.
* Activity Concentration.
Data Table (Concise)