Need help with a Biology HW Question Please

[tr59210]

I have a homework assignment that due and I'm stumped on question 5. I don't know of an easy way to screen 5000 bacteria easily. I know that you can do manually, but that'll take too long. The question seems to be hinting that there's a way to screen the bacteria quickly, as per the last line. Any help would be great. I have an idea of how to do it, but I want some input too. Thank you so much, here's the question:

 

5. P.aeruginosa requires quorum sensing for virulence, and as we discussed inclass, there is significant interest in identifying small molecules thatinhibit quorum sensing. It has been proposed that potential sources of quorumsensing inhibitors are soil bacteria. Design a screen to identify soil bacteriathat produce extracellular molecules that inhibit P. aeruginosa quorum sensing. Consider that you must screen atleast 5000 soil bacteria, and you will be doing all of the work yourself. (10points)

 

 

 

This is from my Metabolism and Biochemistry of Microorganisms class here at UT austin.

[John Cuthber]

Why isn't this in the homework section?

Perhaps more importantly, why have you posted it twice?

[tr59210]

I'm sorry, I didn't know that I had posted it twice and I'll go the HW section right away. Sorry for any inconvenience

[ecoli]

My first thought is some sort of competition experiment.. through all 5000 into a culture or model system and let natural selection enrich for the producers of quorum-inhibitors. I can't think of a way to do this with P. aeruginosa that would be specifically selective though.

[tr59210]

well i was thinking the same thing.

 

Since in nature they all grow together in soil, why not throw the soil bacteria onto a petri plate and see which form colonies (not all 5000 bacteria will grow under laboratory conditions. only about .5-1% of all known bacteria are culturable under laboratory conditions). Then take those colonies and put them in some type of defined liquid culture (separately, one species/strain per culture) and let them grow there. Then spin those cultures down and take the supernatant from each culture and set it aside for use later.

 

Then take P. aeruginosa (assuming you know which genes cause virulence) and make a mutant strain of it by latching a reporter such as GFP to one or more of those virulence genes that are controlled by quorum sensing. Then let the bacteria grow until in reaches the correct population density for quorum sensing to take effect and at that point, you should see some Green under UV light if you were to look at the colonies/biofilm/whatever (because at this point, the bacteria would be turning on those virulence genes you attached GFP to).

 

At this point, you can introduce the supernatants collected from step one (one by one into separate mutant P. aeruginosa cultures), and if they contain a quorum sensing inhibitor, you should see a gradual reduction in GFP (green light under UV) until it's all gone. This will tell you whether or not the supernatant contained a inhibitor and you can trace it back to the culture you got it from.

Edited May 3, 2012 by tr59210

[ecoli]

the problem I see with that method is that it requires you make 5000 separate cultures... which seems to be the thing you want to avoid.

[tr59210]

the problem I see with that method is that it requires you make 5000 separate cultures... which seems to be the thing you want to avoid.

 

no, because in the beginning I said that only .5-1% of bacteria are able to grow under lab conditions, so not ALL 5000 will grow. Only a few.

[Bioc]

But how can you know that the remaining 99% are not producing molecules that inhibit quorum sensing?