Dandekar & Chandler:AHL Extraction
|Line 34:||Line 34:|
Revision as of 17:31, 18 October 2013
N-Acyl homoserine lactone (AHL) Extraction
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.
- Spectrophotometer capable of reading 520 nm
- 10mL test tube
- Sterile Cuvets
- 5ml glass pipet
- Nanopure H20
- 0.2m HCl
- Inoculate overnight culture of reporter in LB + Ap 100 Gen 20 at 37C.
- Subculture to desired volume at low density (~0.05) incubate at 37C with shaking
- At OD600 ~0.2-0.3 induce R expression by adding 0.25% arabinose, continue shaking at 37C
- 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
- 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.
- 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).