Dandekar & Chandler:AHL Extraction

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(Materials Needed)
(Principle)
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===Principle===
===Principle===
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Assay for acyl-HSL detection using E. coli reporters containing pQF50- (QS-controlled lacZ fusion, Ap) and pJN105- (arabinose inducible R gene, Gm) derived plasmids.
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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.
===Materials Needed===
===Materials Needed===

Revision as of 18:36, 18 October 2013

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Contents

N-Acyl homoserine lactone (AHL) Extraction

Principle

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 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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