Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of autoinducers using DH231 (delta cqsS) Measured in Microorganism System Using Plate Reader - 2132-04_Agonist_Dose_DryPowder_Activity_Set2
Assay Overview: This assay assists in identifying the mechanism of action for a particular compound. We will employ a V. cholerae cqsS, luxS double deficient mutant harboring the V. harveyi luxCDABE (luciferase) operon on a plasmid. This V. cholerae strain (called DH231) is naturally dark (Luciferase negative). If a candidate compound acts as an agonist for one of the receptors (say, for example more ..
Keywords:Vibrio cholerae, quorum sensing, CqsS, LuxQ, luciferase
Assay Overview: This assay assists in identifying the mechanism of action for a particular compound. We will employ a V. cholerae cqsS, luxS double deficient mutant harboring the V. harveyi luxCDABE (luciferase) operon on a plasmid. This V. cholerae strain (called DH231) is naturally dark (Luciferase negative). If a candidate compound acts as an agonist for one of the receptors (say, for example CqsS), the mutant deleted for cqsS (i.e., strain DH231) will not make light in response to the candidate molecule. If a compound generates light, it suggests that the compound acts as a agonist of LuxQ
Expected Outcome: Some compounds will generate light in a dose dependent manner suggesting action at the level of the luxQ sensing receptor. Compounds that do not produce light are likely to work as an agonist of the other sensing receptor, CqsS. Either outcome is acceptable for a probe candidate. If a compound is a luxQ agonist, an AC50 of < or = 10 uM is required.
1.For LuxQ agonists: DH231 (V. cholerae delta cqsS delta luxS carrying pBB1 cosmid)
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 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. Use it fresh everytime.
CAI-1 stock: Dissolve CAI-1 in DMSO to 50 mM (10.7 mg/mL), store at -20 degrees C
1.Grow up reporter strain in 50 mL LB/Tet at 30 degrees C 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 30C 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) 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 '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)
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)