Biology Reference
In-Depth Information
3.1.3. AHL Induction/Inhibition Detection
For testing for the presence of inducers or repressors of AHL-based quorum
sensing, the same E. coli monitor strain can be used. To detect inducers, one
can use directly the monitor strain, which does not produce its own AHL signal.
Whereas this monitor strain works well for most AHL molecules, it is not
sensitive to C4-AHLs and another strain must be used, and the method for testing
for the C4-AHLs is described elsewhere (19) . To test for inhibition of signaling,
one must use the monitor strain in the presence of both the test substance and
exogenously added AHL, which in this case is the 3-oxo-6C-AHL.
1. Prepare an overnight culture of the assay strain, E. coli MT102 pJBA132 ( luxR-
P luxI - -RBSII- gfp (ASV)-T 0 -T 1 ) (18) , by growing the organism in 2 mL of ABt
medium, with 6 μg/mL tetracycline at 37°C with shaking (200 rpm).
2a. To test for induction of the AHL-mediated quorum-sensing monitor, the following
morning, add 100 μL of fresh ABt medium to the wells of a 96-well microtiter
plate. To the wells of the top row, add 200 μL total.
2b. To test for the presence of AHL inhibitors in the extract, the following morning,
add 100 μL of fresh ABt medium with 3-oxo-C6-AHL added to a final concen-
tration of 20 n M to the wells of a 96-well microtiter plate. To the wells of the top
row, add 200 μL total volume.
3. Add the extracted material to the first row with the maximum concentration being
equal to twice the MIC as determined above or 200 μg/mL, which ever is lower.
4a. When testing for the presence of AHL inducers, perform doubling dilutions of
the material from the top row to the second to last row, leaving the bottom row
without extract as a control row. To the last row, add 3-oxo-C6-AHL added to a
final concentration of 20 n M as a positive control for induction.
4b. When testing for the presence of AHL inhibitors, perform doubling dilutions of
the material from the top row to the second to last row, leaving the bottom row
without extract as a control row. This last row will serve as a control for maximum
induction in the absence of inhibitor.
5. Dilute the overnight culture 1:100 into fresh medium, with or without antibiotic,
respectively, and aliquot 100 μL into the wells of the 96-well microtiter plate
containing the extracts for testing ( see Note 6 ).
6. Incubate at 37°C and take regular readings for up to 24 h to determine growth yield
(optical density at 600 nm—OD 600 ) and quorum-sensing-regulated Gfp expression
(excitation 475 nm, emission 515 nm) to calculate quorum-sensing induction and
inhibition.
7. Normalize the Gfp expression by dividing the Gfp expression by the OD 600 to
correct for any differences in cell number ( see Note 7 ). The inhibition can also
be visualized using fluorescence microscopy, as shown in Fig. 1 .
3.2. Inhibition of AHL Quorum-Sensing Phenotypes
To verify that a particular extract or compound is active as an inducer or
inhibitor of AHL-based quorum sensing in a relevant pathogen, it is important
 
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