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BioAssay: AID 493096

Quorum Sensing Measured in Microorganism System Using Plate Reader - 2035-02_Inhibitor_DoseNoFile_DryPowder_Activity_Set2

Keywords: Quorum Sensing, Auto-inducer 2 (AI-2), Vibrio harveyi, LuxS, LuxPQ, LuxO, LuxU, luciferase, Luminescence ..more
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 Tested Compounds
 Tested Compounds
All(8)
 
 
Active(7)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(12)
 
 
Active(11)
 
 
Inactive(1)
 
 
AID: 493096
Data Source: Broad Institute (2035-02_Inhibitor_DoseNoFile_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-01-27
Hold-until Date: 2012-01-26
Modify Date: 2012-01-26

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 7
Related Experiments
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AIDNameTypeComment
2106Broad Institute MLPCN Quorum Sensing ProjectSummarydepositor-specified cross reference: Summary assay
2094Plate Read Microorganism-Based Primary HTS to Identify Modulators of the AI-2 Quorum Sensing SystemScreeningsame project related to Summary assay
2723Absorbance Microorganism Dose Retest to Identify Inhibitors of Vibrio harveyiConfirmatorysame project related to Summary assay
2724Luminescence Microorganism-Based Dose Retest to Identify Modulators of the AI-2 Quorum Sensing SystemConfirmatorysame project related to Summary assay
2725Absorbance Microorganism-Based Dose Retest to Identify Inhibitors of Vibrio harveyiConfirmatorysame project related to Summary assay
2726Absorbance Microorganism-Based Retest to Identify Inhibitors of Vibrio harveyiScreeningsame project related to Summary assay
2727Luminescence Microorganism Retest to Identify Inhibitors of the AI-2 Quorum Sensing SystemScreeningsame project related to Summary assay
2728Absorbance Microorganism-Based Retest to Identify Inhibitors of Vibrio harveyiScreeningsame project related to Summary assay
2735Luminescence Microorganism Dose Retest to Identify Inhibitors of the AI-2 Quorum Sensing SystemConfirmatorysame project related to Summary assay
2736Luminescence Microorganism-Based Retest to Identify Modulators of the AI-2 Quorum Sensing SystemScreeningsame project related to Summary assay
434943Absorbance Microorganism Dose Response to Identify Inhibitors of Vibrio harveyiConfirmatorysame project related to Summary assay
434944Luminescence Cell-Free Homogeneous Dose Response to Identify Inhibitors of Lux-SConfirmatorysame project related to Summary assay
493063Quorum Sensing, LuxS, LuxPQ Measured in Microorganism System Using Plate Reader - 2035-01_Inhibitor_DoseNoFile_DryPowder_ActivityConfirmatorysame project related to Summary assay
493072Quorum Sensing, LuxS, LuxPQ Measured in Microorganism System Using Plate Reader - 2035-01_Inhibitor_DoseNoFile_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493075Quorum Sensing, LuxS, LuxPQ Measured in Microorganism System Using Plate Reader - 2035-01_Inhibitor_DoseNoFile_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493097Quorum Sensing Measured in Microorganism System Using Plate Reader - 2035-02_Inhibitor_DoseNoFile_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493101Quorum Sensing, LuxS, LuxPQ Measured in Microorganism System Using Plate Reader - 2035-01_Inhibitor_DoseNoFile_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493103Quorum Sensing Measured in Microorganism System Using Plate Reader - 2035-02_Inhibitor_DoseNoFile_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493104Quorum Sensing Measured in Microorganism System Using Plate Reader - 2035-02_Inhibitor_DoseNoFile_DryPowder_ActivityConfirmatorysame project related to Summary assay
493116Quorum Sensing LuxPQ Measured in Microorganism System Using Plate Reader - 2035-03_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493117Quorum Sensing LuxPQ Measured in Microorganism System Using Plate Reader - 2035-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493118Quorum Sensing LuxPQ Measured in Microorganism System Using Plate Reader - 2035-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
493119Quorum Sensing LuxPQ Measured in Microorganism System Using Plate Reader - 2035-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493167LuxS Measured in Biochemical System Using Plate Reader - 2035-05_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493172LuxS Measured in Biochemical System Using Plate Reader - 2035-05_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
Description:
Keywords: Quorum Sensing, Auto-inducer 2 (AI-2), Vibrio harveyi, LuxS, LuxPQ, LuxO, LuxU, luciferase, Luminescence

Assay Overview: A modified strain of Vibrio harveyi with constitutive on quorum sensing system will be exposed to small molecules identified from the HTS screen used to identify LuxS inhibitors and LuxPQ antagonists. Growth of the organism post exposure will be followed using optical density and disruption of down stream quorum sensing elements will be observed based on decreased luminescent signal.

Expected Outcome: Identification of AI-2 quorum sensing system inhibitors with modes of action which either antagonize the LuxPQ receptor or inhibit LuxS synthase. Such inhibitors should not perturb growth (observed by optical density) nor the constitutive on quorum sensing (observed by luminescence).
Protocol
Protocol:
Add 45 nL 10 mM sample / well to a sterile 384 well black clear bottom assay plate (Greiner microclear). Add 60 uL / well screening culture using a combi dispenser (Thermo) and read OD600 on Envision plate reader (Perkin-Elmer), incubate plate 9 h 30 degrees C in a humid incubator, read OD600 and luminescence on Envision plate reader.
Quorum Sensing Reagents
BB721 Vibrio harveyi (Constitutive On Quorum Sensing )
Overnight Culture
5 mL LM medium plus one colony BB721, 30C 250 rpm
Screening Culture
Overnight Culture diluted to OD600 0.0005 in AB medium
LM Medium (Rich medium for overnights and agar plates)
20 g NaCl (JT Baker, 3624-19)
10 g bactotryptone (BD, 211705)
5 g yeast extract (EMD, 1.03753)
brought to 1 liter, 0.22 u sterile filter
For plates, add 7.5 g agar (BD, 28130) / 500 ml LM autoclave 15 min, plate and flame
AB Medium (Autoinducer Bioassay Medium)
100 ml 10X AB salt
2 g casamino acids (BD, 223050)
10 mL 1 M Phosphate Buffer pH 7
10 mL 0.1 M Arginine
100 uL 1 M Borate
brought to 1 liter, 0.22 u sterile filter
Where:
10X AB salt (3M NaCl, 0.5 M MgSO4)
175 g NaCl (JT Baker, 3624-19)+ 123 g MgSO4 * 7 H20 (Sigma, 230391) brought to 1 liter, 0.22 u sterile filter
1 M Phosphate Buffer pH 7
61.5 mL 1 M K2HPO4 (Sigma, P3786 ) + 38.5 mL 1 M KH2PO4 (Sigma, P0662)
1 M Borate
15.5 g Boric Acid (Sigma, B1934) brought to 500 mL 0.22 u sterile filter
100 mM Arginine
8.71 g Arginine (free base) (Calbiochem, 1820) brought to 500 mL 0.22 u sterile filter
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=304) and positive control wells (PC; n=10) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Organism-based
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.000030uM_(%) (3e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.000060uM_(%) (6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.00012uM_(%) (0.00012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.00026uM_(%) (0.00026μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.0005uM_(%) (0.0005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.001uM_(%) (0.001μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.0021uM_(%) (0.0021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.0042uM_(%) (0.0042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.008uM_(%) (0.008μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.016uM_(%) (0.016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.03uM_(%) (0.03μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.068uM_(%) (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.135uM_(%) (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.26uM_(%) (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.5uM_(%) (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_1uM_(%) (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_2.1uM_(%) (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_4.2uM_(%) (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_8uM_(%) (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_16uM_(%) (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH086452-01

Data Table (Concise)
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