Dandekar & Chandler:AHL Signal Measuring

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(New page: == AHL Signal measuring == In a large test tube containing the sample you wish to test: 1) Add 5mL of ethyl acetate and vortex on high for 30seconds 2) Let the sample sit for 10 minu...)
(AHL Signal measuring)
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== AHL Signal measuring  ==
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In a large test tube containing the sample you wish to test:
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1) Add 5mL of ethyl acetate and vortex on high for 30seconds
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2) Let the sample sit for 10 minutes to allow the two phases to separate out. 
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3) After the the 10 minute rest, pipette off the clear acetate layer only. Be careful to not draw up any of the bottom cell debris layer.  
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4) Deposit the clear acetate layer in a 10mL glass tube with a sealable air tight cap.   
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5) Repeat steps 1-4 and add acetate supernatant to tube from first cycle.  
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== AHL Signal measuring  ==
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6) Place acetate filled tubes into N2 evaporator on medium until all acetate has evaporated off.
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7) Suspend dried signal in 100uL of ethyl acetate, screw sealable cap on tightly, wrap in parafilm and place in -20 freezer.
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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.
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===Principle===
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===Materials Needed===
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* Spectrophotometer capable of reading 520 nm
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* Vortex
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* 10mL test tube
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* Sterile Cuvets
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* 5ml glass pipet
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* Nanopure H20
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* Chloroform
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===Protocol===
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# Inoculate overnight culture of reporter in LB + Ap 100 Gen 20 at 37C.
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# Subculture to desired volume at low density (~0.05) incubate at 37C with shaking
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# At OD600 ~0.2-0.3 induce R expression by adding 0.25% arabinose, continue shaking at 37C
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# At OD600 ~0.5, aliquot 0.5 ml volumes into eppenforf tubes containing the acyl-HSL sample/standard, shake eppendorf tubes for 2 hours at 37C
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# 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 37C shaker. Saran wrap can be easily cut off using scissors at the end of the experiment.  
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# After 2 hours of growth in the presence of acyl-HSL, prepare samples for β-gal assay.  
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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).

Revision as of 18:39, 14 November 2013


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Contents

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 520 nm
  • Vortex
  • 10mL test tube
  • Sterile Cuvets
  • 5ml glass pipet
  • Nanopure H20
  • Chloroform

Protocol

  1. Inoculate overnight culture of reporter in LB + Ap 100 Gen 20 at 37C.
  2. Subculture to desired volume at low density (~0.05) incubate at 37C with shaking
  3. At OD600 ~0.2-0.3 induce R expression by adding 0.25% arabinose, continue shaking at 37C
  4. At OD600 ~0.5, aliquot 0.5 ml volumes into eppenforf tubes containing the acyl-HSL sample/standard, shake eppendorf tubes for 2 hours at 37C
  5. 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 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).

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