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BISC314: Environmental Microbiology

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LAB #4: Quorum sensing - chemical signaling within our community

Many bacteria are able to secrete signals into their environment that they can then utilize to determine their own density. Since bacteria are single-celled organisms, why do you think they would they be interested in knowing how many of them are near? A very well studied example of the quorum sensing system comes from Vibrio fisheri, a bacterium that produces light only at high densities. If you think about it, the light produced by a single bacterium is unlikely to be seen by anyone so Vibrio wait until they have reached a "quorum" before turning on the 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, see this YouTube video of Bonnie Bassler.

Today we will be testing if any of your isolates are secreting Auto-Inducer (AI) into the surrounding media. Specifically, we will be using a strain of bacterium called Chromobacterium violaceum. This organism is gram-negative and normally found in the soil. It produces a very strong purple pigment (hence the name) in response to levels of AI. We will also use a violacein-negative, mini-Tn5 mutant of C. violaceum (CV026) which can produce pigment in response to the AI from other bacteria but can no longer secrete its own AI - this will be our biosensor.

1. Label your agar plates. You will draw a line down the middle of the plate and write CV026 on one side and your isolate code on the other side. Also label a plate for our positive control, the parent strain to CV026: Chromobacterium violaceum ATCC 12472, also known as CV017.
2. Flame your loop. Use your loops to dip into an overnight culture of CV026. Streak 1/2 of an agar plate with this organism (the side with the CV026 label).
3. Flame your loop. Grab a relatively large amount of your isolates and streak them near to the CV026 streaks. Re-flame your loop.
4. Dip the loop into an overnight culture of ATCC 12472 and streak it near to the CV026 streak.
5. Place your plates upside down till next week. If your isolates are producing AI sensed by CV026, you will see a purple pigment.

Recreating your cheese rind -- isolates to community

Today we will also be attempting to recreate the rinds found on your cheese by inoculating a fresh cheese (chevre) with your isolates in combination. You can set up three different experiments and observe what the rinds look like over the next few weeks. Choose three different combinations of isolates or those from different parts of your cheese.

1. Label an empty petri dish with the experimental condition.
2. Label a 1.5 ml microcentrifuge tube your isolate designation and add 200 ul of water to the tube.
3. Flame your loop. Use the loop to take half of an isolate patch and resuspend it in the microcentrifuge tube.
4. Repeat steps 2 and 3 for each of your isolates.
5. Label a 1.5 ml microcentrifuge tube for each experimental condition.
6. Using a micropipette, take 50 ul from each isolate to mix into the experimental condition. For example, if my experimental condition #1 was isolates 4-6, I'd take 50 ul of isolate 4, 50 ul of isolate 5 and 50 ul of isolate 6 in one tube labeled condition #1
. 7. Make sure your isolates are well mixed in the tube.
8. Take a slice of the chevre and place it into the petri dish.
9. Using a micropipette, take 200 ul of your experimental mix and spot it onto the top of the chevre.
10. Close the lid on the petri dish and let the cheese age. We'll keep an eye on them week by week.