Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of autoinducers using WN1103 (delta luxQ) Measured in Microorganism System Using Plate Reader - 2132-05_Agonist_Dose_DryPowder_Activity_Set2
Assay Overview:We will employ a V. cholerae delta luxQ mutant harboring the V. harveyi luxCDABE (luciferase) operon on a plasmid. This V. cholerae strain (called WN1103) is dark (Luciferase negative) in the absence of exogenous agonist and can not produce any CAI-1. Unlike the mutant deleted for cqsS (strain DH231), a candidate CqsS receptor agonist compound will induce light production in WN1103 and rule out activity through the luxQ receptor. ..more
BioActive Compounds: 17
Keywords:Vibrio cholerae, quorum sensing
Assay Overview:We will employ a V. cholerae delta luxQ mutant harboring the V. harveyi luxCDABE (luciferase) operon on a plasmid. This V. cholerae strain (called WN1103) is dark (Luciferase negative) in the absence of exogenous agonist and can not produce any CAI-1. Unlike the mutant deleted for cqsS (strain DH231), a candidate CqsS receptor agonist compound will induce light production in WN1103 and rule out activity through the luxQ receptor.
Expected Outcome: If a compound acts as an agonist of cqsS, luciferase will be generated and light will be emitted. Any outcome will determine mechanism of action and so both agonists and compounds with no activity are acceptable as probe candidates.
Vibrio cholerae quorum-sensing modulator bioassay
1.For CqsS agonists: WN1103 (V. cholerae delta cqsA delta luxQ carrying pBB1 cosmid)
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.
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) and positive control wells (PC; n=20) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
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.
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.
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)
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.
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.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
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
Assay Format: Organism-based
Assay Format: Organism-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)