Quorum Sensing Measured in Microorganism System Using Plate Reader - 2035-02_Inhibitor_DoseNoFile_DryPowder_Activity_Set3
Keywords: Quorum Sensing, Auto-inducer 2 (AI-2), Vibrio harveyi, LuxS, LuxPQ, LuxO, LuxU, luciferase, Luminescence ..more
BioActive Compounds: 9
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).
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 )
5 mL LM medium plus one colony BB721, 30C 250 rpm
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
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
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) and positive control wells (PC; n=9) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing 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 (10)(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): 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)
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.
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)