Dandekar & Chandler:AHL Signal Measuring: Difference between revisions
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===Materials Needed=== | ===Materials Needed=== | ||
* Spectrophotometer capable of reading | * Spectrophotometer capable of reading 600 nm | ||
* Plate reader | |||
* Vortex | * Vortex | ||
* 10mL test tube | * 10mL test tube(s) | ||
* eppendorf tubes (or chloroform resistant 96well plate) | |||
* Sterile Cuvets | * Sterile Cuvets | ||
* | * Glass vial | ||
* Nanopure H20 | * Nanopure H20 | ||
* Chloroform | * Chloroform | ||
* β-gal assay reagents | |||
===Protocol=== | ===Protocol=== |
Revision as of 16:01, 22 January 2014
AHL Signal measuring
Assay for acyl-HSL detection using E. coli reporters containing pQF50- (QS-controlled lacZ fusion, Ampicillin) and pJN105- (arabinose inducible R gene, gentamicin) derived plasmids.
Principle
Materials Needed
- Spectrophotometer capable of reading 600 nm
- Plate reader
- Vortex
- 10mL test tube(s)
- eppendorf tubes (or chloroform resistant 96well plate)
- Sterile Cuvets
- Glass vial
- Nanopure H20
- Chloroform
- β-gal assay reagents
Protocol
> Inoculate overnight culture of reporter in LB plus respective antibiotics (10 μL Amp, 10μL 100 Gen 20 for pSC11 pJ105L / 10μL Amp 100 for pECP61.5) at 37C.
3OC12·HSL via pSC11 pJ105L
- Calculate OD600 of overnight reporter strain + antibiotics
- Subculture to desired volume at low density (~0.05) into 10mL LB + 10 μL Amp, 10μL 100 Gen 20 incubate at 37°C with shaking
- Add 10μL of ethyl acetate concentrated signal to a small glass vial and allow to evaporate in a well ventilated area
- At OD600 ~0.2-0.3 induce R expression by adding 0.25% arabinose (25μL in 10mL), continue shaking at 37°C
- During this shaking period label chloroform-resistant tubes or 96 well plate with sample names and dilutions For ease of handling large numbers of eppendorf tubes, we load them in a rack, wrap saran wrap around the tubes + rack, secure the ends with packing tape, and shake the entire rack in the 37°C shaker. Saran wrap can be easily cut off using scissors at the end of the experiment.
- At OD600 ~0.5, aliquot 100μL into the vial containing (now) dried signal, and appropriate volumes for tubes containing NOT containing the acyl-HSL sample/standard
- Perform desired dilutions across the samples
- Shake eppendorf tubes for 2 hours at 37°C
- After 2 hours of growth add 10% (10μL in 100μL) chloroform to stop growth and lyse cells)
- Shake samples for 30 seconds and let them sit for 10 minutes at room temp.
- Take the top most 10μL from each sample and add it to a corresponding well of an Optiplate™ 96 well plate
- Make a 1:100 mixture of Tropix® Galacton-Plus and Tropix® Galacto Reaction Buffer Diluent and add 70μL to each well containing a sample.
- Place the plate in a dark place at room temp for one hour (a seldom opened drawer works well)
- After one hour dark sit, add 100μL of Tropix Accelerator and read on plate reader. (luminescence, 1000ms, 3 second shake between reads)
C4·HSL via pECP61.5
- Add 10μL of ethyl acetate concentrated signal to a small glass vial and allow to evaporate in a well ventilated area
- Calculate OD600 of overnight reporter strain + antibiotic
- Label chloroform-resistant tubes or 96 well plate with sample names and dilutions For ease of handling large numbers of eppendorf tubes, we load them in a rack, wrap saran wrap around the tubes + rack
- Subculture to desired volume at low density (~0.01) into 10mL LB + 10 μL Amp + 10μL IPTG
- Add
- Secure the ends with packing tape, and shake the entire rack in the 37C shaker. Saran wrap can be easily cut off using scissors at the end of the experiment.
- After 2 hours of growth in the presence of acyl-HSL, prepare samples for β-gal assay.
We lyse cells with 10% vol (50 µl) of chloroform followed by 5 sec vortex and 5 min incubation at RT. 10 µl are then assayed for β-gal using the Tropix kit/luminescence linked detection (10 µl sample, add 70 µl of substrate dilute 1:100 in diluent buffer, incubate 1 hour RT in dark, add 100 µl of accelerator, read luminescence in Tecan plate reader).