Field of Science

Phage plans - let's put natural selection to work!

I have a two-pronged plan to get a phage strain that gives good enough plaques for my GTA-as-vaccine experiments.

I obtained reasonable titers of two phages, 'Titan' and 'Saxon'.  I'll invest a couple of weeks to see if I can get better and more reproducible plaques with either of these.  The genome sequences of these phages are not closely related.

First, improve the plaquing conditions:  The researcher who isolated the phages recommends using for the lawn cells that have been grown photosynthetically to a high density,  He also suggested trying a lower top-agar concentration.  I'll play around with these and other variables to see if I can get better plaques.

Second, use artificial selection to get a better strain of phage:  I'll pick the few best-looking plaques of each of my two phages and plate the phage they contain in new lawns.  From those new lawns I'll again pick the best-looking plaques, and plate their phage in new lawns.  Etc.  Maybe I'll introduce a bit of UV mutagenesis along the way.

The first step will be to make fresh lysates of these phages.  The lawns I made before are too old, so I'll grow up some cells for lawns today and tomorrow I'll retiter the lysates.  On Friday I can pick plaques from these lawns and make plate lysates.  If there's a plate with near-confluent plaques I can use it directly to make a plate lysate.  (10^7 or 10^8 pfu/ml, and I have maybe 5 µl so at best I can get plates with 5 x 10^4 or 5 x 10^5 plaques.  The latter might be enough to get a good lysate.  There are small volumes (50-100 µl?) of the original lysates in the lab upstairs, so maybe I'll sue these.  Or Maybe I should save these until I've improved the plaquing conditions.

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