Friday, April 26, 2013

Illustrating Antibiotic Action for Undergraduates!

In my freshman biology course here at the University of Puget Sound, I think long and hard about ways to show students connections between classroom material and everyday well as straightforward ways to illustrate concepts that students may not find obvious.  Which brings us to today's post.

In my "Unity of Life" course, I need to cover a lot of conceptual ground, with a limited amount of time in a one semester courseDuring the laboratory sessions associated with this class, the students carry out a simple transformation experiment.  Using a very simple "single colony transformation" protocol with E. coli, and the infamous (in education) pGLO plasmid, the students plate onto LB+ampicillin (ssssh...also plus L-arabinose).  Successful transformants should grow on the antibiotic containing medium, and fluoresce under ultraviolet light.  And indeed they do:  

I like the hand-held UV lamps to be found here, which are cheap and work very well.

The pGLO plasmid carries ampicillin resistance (mediated by beta-lactamase), and an interesting fusion between the arabinose operon of E. coli and green fluorescence protein (or GFP).

I reinforce to my students the facdt that the additional DNA containing genes (the plasmid), when expressed by the bacterial cell, will alter the observed phenotype.  This seems quite simple to folks with a lot of biology under their belts, but I have found that this idea and related concepts need reinforcement:  added genes, when expressed in a bacterial cell, can alter the visual phenotype.

Then I wait for four or five days.  The nice big colonies (due to transformation by pGLO) are now surrounded by small "satellite colonies," as seen here:

Yes, this is the reason that many investigators don't care for ampicillin selection in bacteria.  I usually have students pick a "large" colony and a "small" colony and restreak them on LB+ampicillin plates.  Lo and behold, the small colonies do not grow, and the large ones do.

Now comes the fun part.  The same plate you see above, illuminated by UV light:

That's right!  The large colonies fluoresce brightly under UV light, because they have pGLO in action within their cytoplasm.  The beta-lactamase made by these cells (its gene also carried by pGLO)  has been pumped out of the succesful colony, depleting and destroying the ampicillin present surrounding the transformed colony.  Thus, the satellite colonies, lacking pGLO are both ampicillin sensitive and do not glow under UV light.

This series of photographs and explanations underscore several important points:
  • Only a small number of bacterial cells in this experiment have been transformed by pGLO.
  • Ampicillin clearly does not kill untransformed cells.  It is in fact bacteriostatic, and not bacteriocidalThe untransformed cells are inhibited and just sit there glumly.  Once the ampicillin in the medium is destroyed, the patient non-growing cells will begin to grow! 
  • pGLO carries two genes of importance: ampicillin resistance (beta-lactamase) and GFP.  A bacterial cell receiving this plasmid will express both, and change its phenotype accordingly.
This is all quite elementary to many readers, I am very aware.  But I believe that our students need to start somewhere, and my feeling is that this part of the lab will be remembered later.  And the knowledge gained will help the students as the information (and the techniques) become more and more complicated! 

I really enjoyed tinkering with my digital camera and taking photographs for this blog entry.  Sometimes, the results are even somewhat artistic, like this one from another satellite-laden transformation plate.

Art appears in many places!

1 comment:

  1. Spectacular photo! and thanks because I didn't know why the colonies grow that maner. I make clonning and often i have a lot of satellite colonies. Now, I know why.


I am happy to hear your comments and suggestions. I hope to avoid spammage. We shall see how that works out!