M465:Quorum Sensing

Quorum sensing - chemical signaling within our community

Many bacteria are able to secrete signals into their environment to sense their density. Since bacteria are single-celled organisms, 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 any of your isolates are secreting an 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 isolates 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.

Strains used:

Vibrio harveyi BB170 TL26: sensor strain - can respond to AI-2; Vibrio harveyi BB721: positive control - always produces AI-2; Vibrio harveyi BB120: wild type - can both produce and respond to AI-2

Day 1: Start overnight broth cultures of your isolates Grow overnight culture (~14 hours) of TL26, BB721, and BB120 in 5 mL AB broth at 30C (This has been done for you.)

Day 2: Dilute the overnight culture of BB170 culture 1:1000 in fresh AB broth Transfer broth culture of isolates to 1.5 mL microcentrifuge tube and spin down broth cultures of isolates Remove supernatant to new 1.5 mL microcentrifuge tube Transfer broth culture of BB721 and/or BB120 to 1.5 mL microcentrifuge tube and spin down broth cultures Remove supernatant to new 1.5 mL microcentrifuge tube

In triplicate to a black 96 well microtiter plate add: 10 uL of cell free supernatant of your isolate and 90 uL of BB170 1:1000 dilution, repeat with each isolate Negative control: 10 uL of sterile broth and 90 uL of BB170 1:1000 dilution, repeat with each broth type Cell control: 10 uL of sterile H2O and 90 uL of BB170 1:1000 dilution Positive control: 10 uL of cell free supernatant of BB721 or BB120 and 90 uL of BB170 1:1000 dilution

Incubute the plate at 30C in a shaker, after 8 hours measure bioluminescence and OD600