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BioAssay: AID 588436

Cholera Quorum: HTS for inducers of light production in the absence ofautoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_SinglePoint_HTS_Activity

Assay Overview: A modified strain of Vibrio cholerae used in this assay uses light production to signal quorum sensing. Vibrio cholerae is not naturally bioluminescent but the closely related species, Vibrio harveyi, produces light when the population is at a high density (i.e. a quorum is sensed). In the V. cholerae BH1578 strain, the V. harveyi luxCDABE (luciferase) operon was introduced as a more ..
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 Tested Compounds
 Tested Compounds
All(349940)
 
 
Active(553)
 
 
Inactive(349386)
 
 
Inconclusive(1)
 
 
 Tested Substances
 Tested Substances
All(350317)
 
 
Active(553)
 
 
Inactive(349763)
 
 
Inconclusive(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 588436
Data Source: Broad Institute (2132-01_Agonist_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-04

Data Table ( Complete ):           Active    All
BioActive Compounds: 553
Depositor Specified Assays
AIDNameTypeComment
588521Broad Institute Quorum Sensing in Vibrio cholera: CqsS/LuxQ Agonist Probe Projectsummary
652219Quorum 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_Activityconfirmatory
652239Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Inhibitor_Dose_DryPowder_Activityconfirmatory
686945Broad Institute Quorum Sensing in Vibrio cholera: LuxO Inhibitor Probe Projectsummary
Description:
Keywords: V. cholerae, cqsA, luxS mutant, quorum sensing, luminescence
Assay Overview: A modified strain of Vibrio cholerae used in this assay uses light production to signal quorum sensing. Vibrio cholerae is not naturally bioluminescent but the closely related species, Vibrio harveyi, produces light when the population is at a high density (i.e. a quorum is sensed). In the V. cholerae BH1578 strain, the V. harveyi luxCDABE (luciferase) operon was introduced as a cosmid and the operon is activated by endogenous mechanisms to elicit light upon reaching a quorum. In addition, the BH1578 strain is a cqsA, luxS double mutant that lacks both autoinducer synthases (CAI-1 and AI-2). BH1578 does not generate light in the absence of exogenous autoinducers but bioluminescence can be stimulated up to 10,000-fold by adding 1 uM (saturating) CAI-1. Bacteria are plated into 384 well plates and compound is added by the pin transfer method. CAI-1 will be used as the positive control. In addition to luminescence, the confluency of each well is measured at an absorbance of 600 nM.

Expected Outcome: Molecules that induce light production in BH1578 in the absence of exogenous autoinducers will register as hits in this assay. As a reminder of the rationale for the screen, the accumulation of autoinducers normally represses virulence factor expression and biofilm formation in V. cholerae.
Protocol

Vibrio cholerae quorum-sensing modulator bioassay
Reporter strain:
1.BH1578 (V. cholerae AcqsA AluxQ carrying pBB1 cosmid, which contains the V. harveyi luxCDABE luciferase operon)
Materials:
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
Tetracycline (10 mg/mL): Dissolve 10 mg tetracycline in 1 mL 100% ethanol, store at -20 degrees C, protect from light
LB/tet: add 1 mL tetracycline (10 mg/mL) to 1L of LB medium. Final concentration of tetracycline is 10 microg/mL. Prepare it fresh on a daily basis.
CAI-1 stock: Dissolve CAI-1 in DMSO to 50 mM (10.7 mg/mL), store at -20 degrees C
Procedures:
1.Grow up the BH1578 reporter strain in 100 mL LB/Tet at 30 oC for >16 hours with shaking (200 rpm). The final OD600 of each culture should be > 3.0
2.Dilute the culture to an OD600 of 0.9 with LB/Tet, mix well. (Note: avoid biofilm aggregates in the culture, a low speed centrifugation (200 rpm for 1 min) should remove most aggregates)
3.Add 20 uL of LB-Tet per 384 well assay plate with the Thermo Combi fluid dispenser. Add 150 nL of compound per well.
4.Dispense 10 microL of diluted culture into each well of a 384 well plate (Black with clear bottom Greiner 781096 plates). Compounds are screened at 20 microM final concentration. 1 uM CAI-1 was used as the positive control.
5.Incubate the plates at 30 degrees C for 6 hours.
5.Measure bioluminescence (USLum(384)) and OD600 in a Perkin-Elmer Envison Multilabel Reader
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 30.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 50.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

tSamples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

NOTE: Raw signal values for inactive compounds were outside the linear detection range of the plate reader; therefore, no REPRODUCIBILITY_COSINE_TRANSFORM was calculated for inactive compounds.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid. Not calculated for inactive compounds, see note in comments.
Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_18.7uM_(%) (18.7μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_18.7uM_(%) (18.7μM**)The calculated activity for the indicated sampleFloat%
5REPLICATE_C_ACTIVITY_SCORE_18.7uM_(%) (18.7μM**)The calculated activity for the indicated sampleFloat%
6REPLICATE_D_ACTIVITY_SCORE_18.7uM_(%) (18.7μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: R03 MH094166-01

Data Table (Concise)
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