The Viralcoaster: My highs and lows

Wow, we are at the end of a semester already!  I don’t know about the rest of my class, but I have been left wanting.  Despite all the time, effort and experience that has been packed into these last 14 weeks, all I want to do is keep coming into the lab and playing with my phage.

There’s something about this ‘choose your own adventure’ styled course that has gotten its hooks into me (or perhaps that’s just a wayward podovirus [1.]).  One of my absolute favourite parts of this journey has been designing or modifying my own experiments to characterise my phage, and I just don’t want to stop!

My hunt for phage began with a stubborn determination to shirk the easy route, the obvious choices.  For the first three weeks I was tramping through our beautiful (and muddy) native bush, picking my way across equine graveyards, and coming perilously close to falling into multiple lakes, all in the quest to conquer the challenge I set for myself – finding a phage somewhere other than a compost bin.  I should have paid more attention to the obvious facts: bacteria love compost, and phages love bacteria.

Eventually I caved, and I get a kind of sentimental warmth from knowing that my phage came from the location-that-shall-not-be-named of my darling mum, an avid gardener.  Not only did my search end somewhere symbolic of my mother, but the name I chose for my precious virus came from my father’s mother.  Both women are actually named Colleen, but my Nana was always known by her middle name, Dulcie.  We lost her when I was a teenager, and I think it’s nice that I’ve taken this opportunity to connect to her a little more in my adult life.  She wouldn’t have given a toss about bacteriophages, but she would have appreciated the thought.

nana photo

I know what you’re thinking: the resemblance is uncanny.

 

After finally hitting the jackpot with plaque assays that I tend to compare to swiss cheese, I proceeded to isolate and process my phage samples ready for the next stage of our adventure.  I started off isolating two phages, before abandoning one to focus my attention on Dulcie.

plate photo for blog

From discovery…                                  to isolation…                                      to amplification

 

Once I had isolated my phage and achieved a high-titer lysate to work with, I proceeded to extract dear Dulcie’s DNA and take a look at it using restriction enzyme digests and gel electrophoresis.  I was pretty stoked with the concentration of DNA in my fourth and fifth rounds of extraction, in which I used a spun-down sample of my lysate.  Those concentrated samples yielded an average of 250 µg of DNA per mL, six times more concentrated than previous extractions.  I had more DNA than I knew what to do with!

My first attempt at gel electrophoresis didn’t go so well, with some degradation of the DNA occurring due to over-enthusiastic nucleases [2].  Use of EDTA solved that smudgy little problem, and the next gel I ran had me dancing on the spot when I saw the photo:

copy for blog Dulcie gel electrophoresis labelled

Dulcie’s DNA following restriction enzyme digest and gel electrophoresis

 

From there, life in phage lab has been all about figuring out which experiments I wanted to run to distinguish Dulcie from other phages, and waiting for the much-anticipated trip to view ‘her’ using transmission electron microscopy.  Memories of that visit will have me smiling for the rest of the year; not solely due to the experience, but also the recollection of four grown-ass independent young women reduced to squeals, ‘ooh’s ‘aah’s and ‘whoa’s with each image that popped up on the monitor.

Created by Digital Micrograph, Gatan Inc.

Can you blame us?  Lots of little Dulcies is pretty phagin’ cool!

In the remaining weeks of semester, I have been putting Dulcie through her paces.  We’ve been testing whether she can switch to the lysogenic cycle (still not sure on that one, our tests were inconclusive), how hot she likes her spa pool (apparently 50ºC is just the ticket), and also just how viral a virus she is.

We are just about to send our DNA samples away to be sequenced under the SEA-PHAGES program, so the next exciting chapter will be finding out what her genome looks like.  Until then, I’ll be spending my time gazing wistfully out the window and daydreaming about that one time, in phage lab…

References:

[1]  Aksyuk, A. A., Bowman, V. D., Kaufmann, B., Fields, C., Klose, T., Holdaway, H. A., . . . Rossmann, M. G. (2012). Structural investigations of a Podoviridae streptococcus phage C1, implications for the mechanism of viral entry. Proceedings of the National Academy of Sciences, 109(35), 14001-14006.

[2]  Nishino, T., & Morikawa, K. (2002). Structure and function of nucleases in DNA repair: shape, grip and blade of the DNA scissors. Oncogene, 21(58), 9022-9032.

 

Advertisements

About jcturnbullnz

BSc Genetics student and novice phage hunter
This entry was posted in Uncategorized. Bookmark the permalink.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s