Field of Science

Phage phrustration

Aacckk!  I've spent more than a month trying to get decent R. capsulatus phage plaques on R. capsulatus lawns.  Still no consistent success.  In one experiment I had much better plaques on cells of strain DE442 (a GTA overproducer), but that did not replicate.  I suspect that non-tiny plaques depend on exactly the right balance of the cells' physiological state, their density, the agar concentration, the culture medium, and other factors I haven't attempted to vary.

Yesterday I was almost ready to do a UV-irradiation experiment to generate mutant phages that make larger plaques, starting with two lysates I'd grown up from single plaques that were much larger than the rest.  But the dilution-series plates I was titering these lysates on grew up with very similar numbers of (mostly tiny) plaques, suggesting that phage contamination had crept into lysates with unexpectedly very low titers. 

This week I've gotten a couple of good suggestions from visitors:

The first visitor suggested that I give up on the characterized/sequenced phages I've been working with and just isolate a better-behaved phage from R. capsulatus's natural environment.  I'd have to learn how to do this (get water

Another visitor suggested that maybe the problem is the correctness of my hypothesis about GTA transduction of phage DNA leading to CRISPR-mediated phage immunity in the GTA recipient.  That is, maybe the first cells to get infected in my lawns produce so many phage-DNA-carrying GTA particles that many of the neighbouring cells that would otherwise be lysed by the phage become immune to the phage before the plaque can form.

There's a simple way to test this - see if the phage form better plaques on a strain that doesn't produce GTA.  So tomorrow I'm getting some GTA mutants from the guy upstairs.

No comments:

Post a Comment

Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="">FoS</a> = FoS