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BioAssay: AID 624269

Quorum LuxO: Assay for inducers of light production in the absence of#autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_Agonist_Dose_DryPowder_Activity

Assay Overview: This assay using Vibrio cholerae strain BH1651(luxOD47E) will help to define the mechanism of action for each compound. LuxOD47E is a constitutive allele of LuxO that locks the protein into a form mimicking LuxO~P. The LuxO~P mimetic activates transcription of the genes encoding the small regulatory RNAs (Qrr1-4). This, in turn prevents translation of HapR which is required for more ..
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AID: 624269
Data Source: Broad Institute (2132-03_Agonist_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-06-06
Hold-until Date: 2013-06-06
Modify Date: 2013-06-06

Data Table ( Complete ):           Active    All
BioActive Compounds: 3
Depositor Specified Assays
AIDNameTypeComment
588521Broad Institute Quorum Sensing in Vibrio cholera: CqsS/LuxQ Agonist Probe ProjectsummarySummary assay
686945Broad Institute Quorum Sensing in Vibrio cholera: LuxO Inhibitor Probe Projectsummary
Description:
Keywords: Vibrio cholerae, quorum sensing, luxO, luciferase


Assay Overview: This assay using Vibrio cholerae strain BH1651(luxOD47E) will help to define the mechanism of action for each compound. LuxOD47E is a constitutive allele of LuxO that locks the protein into a form mimicking LuxO~P. The LuxO~P mimetic activates transcription of the genes encoding the small regulatory RNAs (Qrr1-4). This, in turn prevents translation of HapR which is required for activation of luciferase. Therefore, this strain is naturally luciferase negative and only if a compound acts on luxO or downstream of luxO will there be light production, measured as an increase in signal.


Expected Outcome: Compounds that act on LuxO or downstream of LuxO (e.g. on Hfq) will induce light production in BH1651 and will lead to an increase in signal (i.e. light production). Since this assay is used to define mechanism of action, any outcome is acceptable for a probe candidate.
Protocol
Reporter strain:
1.For LuxO inhibitors: BH1651 (V. cholerae luxOD47E carrying pBB1 cosmid)
Materials:
LB Medium: Dissolve 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 oC, 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. Use it fresh everytime.
CAI-1 stock: Dissolve CAI-1 in DMSO to 50 mM (10.7 mg/mL), store at -20 oC
Procedures:
1.Grow up reporter strain in 50 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 culture to a final OD600 of 0.3. (Note: avoid biofilm aggregates in the culture, a low speed centrifugation (200 rpm for 1 min) should remove most aggregates)
3.Dispense 20 uL of LB/Tet per well of the 384 well plate (Black with clear bottom, such as Greiner 781096 plates).
4.Pin transfer compound (100 nL volume).
5.Dispense 10 microL of diluted culture into each well of a 384 well plate
6.Incubate the plates at 30 oC without shaking for 6 hours.
7.Measure bioluminescence (USLum(384)) and OD600 in a Perkin-Elmer EnVision Multilabel Plate Reader.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing 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 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): absACnn, the concentration at which the curve crosses threshold 40.0
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 >= 30% but <= 70%, 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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AbsAC40_Qualifier<, =, or >String
2AbsAC40_uM*The concentration at which the fitted curve passes activity threshold 40.FloatμM
3pAbsAC40_MEqual to -1*log10(AbsAC40).Float
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_0.000060uM_(%) (6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.00012uM_(%) (0.00012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.00026uM_(%) (0.00026μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.0005uM_(%) (0.0005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.001uM_(%) (0.001μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.0021uM_(%) (0.0021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.0042uM_(%) (0.0042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.008uM_(%) (0.008μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.016uM_(%) (0.016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.03uM_(%) (0.03μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.068uM_(%) (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.135uM_(%) (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.26uM_(%) (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.5uM_(%) (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_1uM_(%) (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_2.1uM_(%) (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_4.2uM_(%) (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_8uM_(%) (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_16uM_(%) (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_35uM_(%) (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

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

Data Table (Concise)
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