Finishing Biolog Assays
Today you will take the final measurements for your Biolog Ecoplates using the spectrophotometer. Afterwards, we will discuss how to analyze these data, and what kinds of questions you might ask with these data using the class dataset.
1. Take your plates out of the incubator.
2. Take a reading at A590 nm using the BioTek Synergy H1 384 plate reader (your instructors will show you how)
3. Discard your plate in the biohazardous waste containers.
Carbon source utilization pattern:
How do we analyze our data? You could plot on a bar graph the average A590 nm absorbance of the three replicates (with error bars) on the final day of data collection on the y axis and the 31 different carbon sources on the x axis in one figure. (Remember that there is no such thing as a negative value for Absorbance so count anything that is less than zero as zero. Why might you seem to have a negative value?). Should you arrange those carbon sources in the order they are on the BIOLOG plate or is there a better way to organize them on your graph to show your main message(s)? What kind of information do you need in the figure legend?
Can you think of other ways to illustrate metabolic diversity of your community?
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. Each of these tubes contains a different type of fly, be sure to keep track!
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.
1. Place all cultures in the metal bins near the door.
2. Place your biolog plates, lysates and tubes in the biohazardous wastes.
3. Place your black bottom plates (with the QS strains) in the 30C incubator
4. Wipe down all surfaces and wash your hands