Many bacteria are able to secrete signals into their environment to sense their density. Since bacteria are single-celled organisms, can you think of why would it be important for them to sense density?
A very well studied example of a quorum sensing system was discovered in Vibrio fisheri, a bacterium that produces light only at high densities. Because the light produced by a single bacterium is unlikely to be detectable, it makes sense to wait until a "quorum" is reached before turning on the expensive metabolic pathway that creates light. In this way, a gene regulatory network is actually controlled by cell density. To hear more about it from another source, visit this YouTube video: Bonnie Bassler.
Today we will be setting up a test to see if your bacterial community found associated with Drosophila is producing Auto-Inducer (AI) into the surrounding media. Specifically, we will be using a strain of bacterium called Vibrio harveyi. This organism is a Gram-negative bioluminescent bacteria that lives in marine environments. It is able to sense AI-2 that might be secreted by your microbes and respond. The response is to produce bioluminescence if enough AI-2 is sensed. We will be using a mutant strain of Vibrio harveyi. This mutant can produce light in response to the AI from other bacteria, but can no longer secrete its own AI - this will be our biosensor.
Vibrio harveyi TL26: sensor strain - can respond to AI-2;
Vibrio harveyi BB721: positive control - always produces AI-2;
Using a Vibrio harveyi reporter strain to detect production of AI-2 by the Drosophila community
1. Dilute the overnight culture of TL26 1:1000 in fresh AB broth (provided in tubes at the instructor's bench). Each test tube contains 5 mL of AB broth - add 5 uL of your TL26 overnight culture.
2. Obtain four 1.5 mL microcentrifuge tubes with Drosophila in 500 uL of PBS and homogenize the flies inside using a sterile pestle (remember to return the contaminated pestles to the front). Each of these tubes contains a different type of fly, be sure to keep track! Do not discard this lysate as it will be used below.
3. Transfer the culture supernatant of BB721 to a 1.5 mL microcentrifuge tube and spin down to remove the microbes. Remove the supernatant and transfer it to new 1.5 mL microcentrifuge tube.
4. In triplicate, to a black 96 well microtiter plate add:
- 10 uL of your Drosophila lysate
- 90 uL of the TL26 1:1000 dilution
repeat with each Drosophila genotype
5. As a negative control, in triplicate, set up wells containing 10 uL of sterile PBS and 90ul of the 1:1000 TL26 dilution.
6. As a positive control, in triplicate, add 10 uL of cell free supernatant of BB721 and 90 uL of TL26 1:1000 dilution.
7. When you are done loading your samples into the plate, seal the plate with a translucent film (provided by your instructor).
8. Incubate the plate in the incubator and after 8 hours measure the bioluminescence and OD600 using the Synergy plate reader. This may mean that you are coming back to this room late tonight! Make sure you take note of the time. Your assay will not work if you do not come back between 7-9 hours after your add your reporter strain to the black plate.
Quorum sensing - setting up our cultures for next time
When we meet on Thursday we will need cultured microbes from your flies. In order to make sure this is prepared, we will be plating a dilution from your flies.
1. Using the lysate created in activity #1 above, make a dilution of your lysates by adding 10 ul to 190 ul of sterile PBS in a sterile, labeled 1.5 mL tube.
2. To each of one agar plate for each media type (MRS and LB) add 100 ul of your lysates and using sterile beads, spread the lysate evenly.
3. Dispose of the beads in the bin at the front of the room and incubate your plates at 30C until next time!
note: you should have two plates at the end of this experiment for each fly genotype, one of MRS and one of LB.
1. Place all cultures in the metal bins near the door.
2. Place your lysates and tubes in the biohazardous wastes.
3. Place all contaminated pestles in the beaker at the front.
4. Place your black bottom plates (with the QS strains) in the 30C incubator
5. Wipe down all surfaces and wash your hands