Last time we met, we tested for the ability of a Vibrio harveyi reporter strain to detect the production of autoinducer-2 by your Drosophila community. Today, we will test for the production of another kind of autoinducer, acyl homoserine lactones, using another reporter strain.
This reporter is an Agrobacterium tumefaciens KYC55 strain that over-expresses the receptor for AHLs, allowing it to detect a variety of AHLs at low concentrations. When this strain detects AHLs, it produces β-galactosidase which breaks down X-gal to produce a soluble blue product.
For this lab, you will use your isolates, grown on agar plates from our last meeting, and overlay them with cells of the reporter strain and X-gal. If the reporter strain detects AHLs from your isolates, the reporter will produce β-galactosidase, break down X-gal, and produce a blue color. If they do not detect AHLs, the overlay of Agrobacterium will remain white.
Agrobacterium tumefaciens KYC55 (pJZ372)(pJZ384)(pJZ410)
pJZ372 (traI-lacZ, AHL responsive reporter fusion), TcR; pJZ384 (PT7-traR, AHL responsive transcription factor), SpR; pJZ410 (T7 RNA Polymerase), GmR
Detecting AHLs produced by Drosophila microbiome members
0. Remove your agar plates from the incubator. These plates contain colonies of isolated microbes from your wild-caught Drosophila.
1. Add 5 mls of 20X AT salts, 5 mls of AT buffer, and 1 ml of 50% glucose to 90 mls of sterile, fully molten 0.6% water agar at 45°C.
2. Mix, and add 200 uls of 20 mg/ml X-gal and 1 ml of prepared the KYC55 cells (provided by your instructor).
3. Gently mix and overlay each of your agar plates with 20-25 mls of the molten agar mixture (this should cover each plate but does not have to be exact).
4. Allow overlays to solidify (~30 min) and incubate overnight at 30°C. Do not invert plates!
5. As a negative control, pour the KYC55 cell mixture over sterile agar plates of the same media type.
6. At the end of this experiment, you should have ten plates - two negative controls (one for LB and one for MRS) and two plates from each type of media for your specific fly genotypes.
1. Wipe down all surfaces.
2. Place all glass culture tubes in the metal racks by the door.
3. Place your agar plates - agar side down - in the incubator.
4.Toss all unused Agrobacterium cultures in the biohazardous waste.
5. Wash your hands