Tuesday, April 22, 2014

Introducing Freshman to Transformation and Gene Regulation

In my current position at the University of Puget Sound, I generally see freshman students in my Biology 111 ("The Unity of Life") course, as well as juniors and seniors in my Biology 350 ("Microbiology") course.  I would prefer to call the latter "Microbial Diversity," because that is the actual thrust and overarching theme of the course, but I have been told that such a title might confuse medical school admissions boards.  Um.

Someday.

Many of my freshman have not had much in the way of biology, and some concepts that are basic to future courses in the major are vital.  Among them are transformation, antibiotic resistance, and gene regulation.

In the laboratory portion of Biology 111, we use the famous pGLO plasmid, which contains a transcriptional fusion between the araBAD promoter and green fluorescence protein (GFP).  Thus, when the araBAD promoter is "active," GFP is made.  And GFP is a wonderful thing for students to observe, due to its lovely color and the fact is is clearly evident with inexpensive UV "flashlights."

The restriction map of pGLO is pretty straightforward.

From:  http://classroom.sdmesa.edu/eschmid/Lab7-B1.jpg
Here is a photo I took of E. coli containing pGLO on a plate containing ampicillin (encoded by the plasmid) and the sugar L-arabinose.


It's interesting to discuss this process with students.  The concept that the bacterium that takes up the plasmid DNA by transformation will then express the genes carried by the plasmid can sometimes take a bit of getting used to for students.  But it is a strong foundation to base other concepts upon.  I often say that the role of an introductory course is to create a "tree" of connected concepts on which branches and leaves can be placed later, in future courses.  So it is with transformation and plasmids and drug resistances.

What I adore showing students is what happens later.  One of the problems with ampicillin resistance in the molecular biology lab is that it is usually mediated by the secreted enzyme beta-lactamase, which cleaves the beta-lactam ring of penicillin-like drugs such as ampicillin.  This in turn means that the concentration of ampicillin surrounding the transformant colonies decreases.  So far, so good.

One of the things that I emphasize to students is that only a small proportion of the bacterial cells can successfully take up and express the plasmid DNA (compared to the cells that do not).  And more importantly, that many antibiotics actually don't kill bacteria...but simply keep them from growing.  Thus, with time, a transformation plate using ampicillin resistance can look like this.


I really am proud of this photo; it looks lovely to me.  

The glowing central colonies (the original transformants, containing pGLO) have degraded much of the ampicillin surrounding them, allowing nontransformed cells to eventually grow.  These are the famous "satellite colonies" that bedevil many molecular biologists who leave their plates out for the weekend before picking transformants!  I have them pick a big colony, streak it out, and show that it both glows on arabinose medium and is ampicillin resistant.  What amazes them is that the satellite colonies (that do not glow) are sensitive to ampicillin when tested the same way!

This is a fabulous concept question for final exams!  It really illustrates the central concepts involved, and involves critical thinking.

Most of the time, people who use pGLO ignore the fact that it is essentially a reporter gene construct:  linking GFP synthesis to the activity of the araBAD promoter of the arabinose operon of E. coli.  

So I have a couple of issues I like to show students, as illustrated from a lecture slide below.
The difference between D and L arabinose seem minor in terms of shape; like the difference between a right and left glove.  Yet the regulatory system does not recognize D-arabinose!  I illustrate this by having students shake hands normally, and then with both of their right hands.  Recognition is shape.  

D and L arabinose are enantiomers of one another, and this is a good way to illustrate that "mirror images" of the same chemical can have quite different effects.  Molecular shape---recognition---is all about that.  And it is highly relevant to organic chemistry and pharmacology, as enantiomers of certain drugs can act quite differently from one another.

This idea is central to my course.  Heck, I even give it a fake name:  stericity.  Molecular shape confers functionality.  Alter that shape, and the functionality is altered, too.

Finally, when discussing gene regulation and operons, the idea of multiple levels of control sometimes confuses students (perhaps not yours; I can only discuss my own experiences in the classroom and laboratory).   So I try to get students to think about the arabinose operon (because of the connection to pGLO, actually).

From:  https://www.bio.cmu.edu/courses/03441/AraGal/AraOverview.jpg
I like to use the pGLO system to illustrate catabolite repression. Illustrations from a textbook are perfectly accurate, but can be confusing to some students.  Getting the students to think about the cell "preferring" to use glucose over arabinose might help.  Here is how textbooks show this "choice" issue.

From:  https://www.bio.cmu.edu/courses/03441/AraGal/AraExpression.jpg
But that can seem confusing.  So I try to take things back to the pGLO system.  Once again, from a lecture slide I made.
Now the material in textbooks about CAP and cAMP and glucose and lactose can make more sense.  First, I try to get students to call CAP (the catabolite activator protein) by another name:  CRP or the cAMP receptor protein.  I show the structures of L-arabinose and glucose, and ask which is easier for a cell to "use" and why they would think so.  Then I discuss the role that cAMP has as a "signal" of glucose availability. 

And sure enough, on the combination plate (glucose + arabinose), GFP is turned off in the presence of glucose.  Over the next week, guess what?  GFP gets turn on and is visible on the combination plate.  It's great to ask the students why (as the glucose is used up, cAMP levels rise, which form a complex with CRP, assisting in activation of the araBAD promoter).

I have also been known to put pGLO into E. coli deletion mutants in the crp and cya (adenylate cyclase) genes...to show the role of that system in gene regulation---putting it all together.

To summarize, it is very possible to use this humble plasmid to illustrate many central concepts in molecular biology and bacterial genetics.  And because of the fluorescence of GFP, it is the kind of thing that students enjoy, and remember.

Now, if only I had similar constructs between araBAD and RFP or YFP.  Maybe someday.  For now, I know that this works well for my freshmen.

Sunday, April 20, 2014

Some "Holiday" Themed Microbiology?

It's Easter Sunday as I write this quick post.  And don't worry:  I'm not going to discuss religious issues.  For many people (especially those of us with children), Easter is much more about high fructose corn syrup.  Right?  Sad but true.

One type of scary yet oddly delicious sugary item for this holiday seasons are the infamous Peeps.  They become all the rage during this time of year, with their near total lack of nutritional value, in addition to colors and textures not normally observed in nature. Peep mania becomes "a thing." Some people get carried away a bit with Peep Dioramas each year. 

But I look at Peeps scientifically.

There are some scientists who "get" the season in the fashion I am describing, and have funny websites like the Institute for Peep Science.  If you haven't been gone to that site, their evidence that smoking and drinking are bad for you, demonstrated with Peeps, is hysterical. 

And there are many, many other sites that explore ersatz-science using our sugary Eastertime friends.  Here is a nice review.  Searching around the internet yields such great stories as the hard, cold facts regarding Peep Jousting.  Or for the space-mad among us, even what happens when you put an evil or misbehaving Peep outside the airlock into vacuum.  

Science of the tasty and humorous variety! 

In that spirit, my lovely and talented wife Jennifer Quinn (not just an authority in mathematics, but a wonderful and creative artist) made me the following video.



Oh, sure.  I should have had her write about agar instead of gelatin (forgive me, Fanny Hesse!).  But it is still awfully funny.

Thank you, Jenny!  You made me smile.  And now, back to that intimidating Tower of Grading...

No matter how (or if) you celebrate this day, I extend my very best microbiological wishes to every reader.

Friday, April 18, 2014

Former Research Students and Their Success Stories, Part I!

At a small liberal arts institution, I don't have postdoctoral students, technicians, or graduate students.  I lack vast infrastructure or much in the way of grant support (and I am pretty much out of money---gulp!).  Nor do I have much time, with teaching taking up most of my hours on campus (and off, given grading and prep time).  I have very little time to reflect and plan and be creative, though I do my best.

What I am very fortunate to have are wonderful undergraduate students.  On this blog, I would like to write about those students from time to time.  After all, a great career after graduation is certainly a fine "outcome" of student education here in Tacoma! And I have many success stories to share. 

One such success story is the inimitable Franny Gilman, who graduated from the University of Puget Sound in 2010.  Franny worked with myself and my colleague Stacey Weiss on the fairly unusual cloacal microbiota of the Striped Plateau Lizard, Sceloporus virgatus, and we had a publication in the journal Symbiosis as a result.

After graduation here in Tacoma, Franny went on to graduate school in the Division of Biological Sciences at the University of Montana.  There, she began work with William Holben's group for her PhD research.  Last summer, Franny went to Greenland to study the microbial ecology of that far-flung place.  Here is her story, in her words, and with her photographs.

Microbial contributions and responses to climate change vastly exceed previous expectations and appear to be substantially impacting both climate change rates and ecosystem responses, yet remain poorly understood. This microbial "black box" needs to be exposed and examined to provide more accurate climate models.  
Specifically, my thesis aims to address the microbial community composition and community function observed in permafrost and the correlation to greenhouse gas emissions measured in two different field sites in Greenland.  My thesis research will involve deployment of molecular analyses including deep 16S rRNA sequencing, metagenomic analyses, comparative metatranscriptomics, and quantitative PCR for key enzymes in controlling methane production and carbon and nitrogen cycles.  Using these techniques will help to develop better predictions as to how microbial communities shift in changing environments in the arctic.  
This past summer I was able to work with collaborators from the Center for Permafrost at the University of Copenhagen (http://cenperm.ku.dk/) and the Geological Survey of Denmark and Greenland (http://www.geus.dk/geuspage-uk.htm) in both Copenhagen and in Greenland.  
I am now back at my home lab at the University of Montana (http://holben-lab.dbs.umt.edu/).
It kind of makes me a little teary to hear how professionally Franny writes about all the wonderful things she is doing in the lab---and yes, out on the glacial ice!  It's true that I wish I had those resources and wonderful instruments to work with, but I am so very proud of Franny and how much she has learned since leaving my classroom and laboratory.

Here are some photographs from Franny's Summer 2013 adventure in Greenland!

Figure 1. This photo was taken outside of the Arctic Station on Disko Island in Greenland in August (http://arktiskstation.ku.dk/english/).

Figure 2.  After a day of soil sampling, two other graduate students and I head back to the station around 9 at night.  This photo was also taken in August.

Figure 3.  Here I am at the CENPERM snow fence field site in June.  
And that last image fits Franny to a "T"...and is how I always think of her.  One of a kind!

I had asked Franny to get me some souvenirs from Greenland. After all, it is not likely that I shall ever travel there!  And she sent me some interesting things, indeed, for my Wunderkammer or Cabinet of Curiosities!

There exists remarkable lichen on some of the volcanic rock in Greenland.  Check out the piece Franny sent me.


I have also heard that Greenland contains some of the oldest rocks on the planet (including evidence of ancient microbial life), and some are quite unusual.  In particular, I have heard that many minerals in Greenland fluoresce under ultraviolet light. And indeed, one of the rocks Franny sent to me does precisely that!

 Sure, I adore the souvenirs---thank you, ma'am!---but I am far, far more excited to hear of the wonderful and interesting things Franny is doing in graduate school.  I hope to add her PhD thesis to my shelf, along with those of my other former undergraduate research students.  It's a awesome thing to look at while I am grading at my desk, and helps me through tough times.

Watching former students succeed and do wondrous things is the best "thank you" I can ever receive.

This is why I have the job I do, warts and all---to have the privilege of working with students like Franny Gilman.  Franny, you have come a long way from when we first met!  I hope to get you to come back to Tacoma to give a seminar about your work soon.

I'm very proud of you, and thanks again for being in my classes, and working in my laboratory.