Dandekar & Chandler:AHL Signal Measuring

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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.

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

1. Calculate OD₆₀₀ of overnight reporter strain + antibiotics
2. 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
3. Add 10μL of ethyl acetate concentrated signal to a small glass vial and allow to evaporate in a well ventilated area
4. At OD₆₀₀ ~0.2-0.3 induce R expression by adding 0.25% arabinose (25μL in 10mL), continue shaking at 37°C
5. 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.
6. 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
7. Perform desired dilutions across the samples
8. Shake eppendorf tubes for 2 hours at 37°C
9. After 2 hours of growth add 10% (10μL in 100μL) chloroform to stop growth and lyse cells)
10. Shake samples for 30 seconds and let them sit for 10 minutes at room temp.
11. Take the top most 10μL from each sample and add it to a corresponding well of an Optiplate™ 96 well plate
12. Make a 1:100 mixture of Tropix® Galacton-Plus and Tropix® Galacto Reaction Buffer Diluent and add 70μL to each well containing a sample.
13. Place the plate in a dark place at room temp for one hour (a seldom opened drawer works well)
14. 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

1. Add 10μL of ethyl acetate concentrated signal to a small glass vial and allow to evaporate in a well ventilated area

1. Calculate OD₆₀₀ of overnight reporter strain + antibiotic
2. 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
3. Subculture to desired volume at low density (~0.01) into 10mL LB + 10 μL Amp + 10μL IPTG
4. Add
5. 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.
6. 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).