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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Not your typical science blog, but an 'open science' research blog. Watch me fumbling my way towards understanding how and why bacteria take up DNA, and getting distracted by other cool questions.
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