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…

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thin lawns, feeble or absent phage

My phage titering gave disappointing results.  Three of the five lysates gave no plaques at all, and the other two gave small indistinct plaques that couldn't be accurately counted or characterized. I took some photos of the plaques I did see.  The top photo is a section of one lawn, with several thousand tiny indistinct plaques.  (The blurry markings are the label on the bottom of the plate.)  The second photo is a closeup  of…

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Titering my lysates

Planning today's work:Titering the phage lysates should be a no-brainer, but it's been a long time since I worked with phage so I'd better think things through before I do it.I have 15 µl of each of 5 phage stocks ('lysates').  The original titers (plaque-forming units/ml, pfu/ml) are written on the tubes - they range from 6x10^5 pfu/ml to 2x10^11 pfu/ml.  But the lysates are probably quite old (maybe 2 years, maybe more), so their…

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Titering my lysates

Planning today's work:Titering the phage lysates should be a no-brainer, but it's been a long time since I worked with phage so I'd better think things through before I do it.I have 15 µl of each of 5 phage stocks ('lysates').  The original titers (plaque-forming units/ml, pfu/ml) are written on the tubes - they range from 6x10^5 pfu/ml to 2x10^11 pfu/ml.  But the lysates are probably quite old (maybe 2 years, maybe more), so their…

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About bacterial lawns and phage plaques

This was going to be a post where I do the planning to titer my new lysates today, but it turned into an explanation of how microbiologists use plaques in lawns of bacteria to study phages.Wait, what's a 'lawn' and what's a 'plaque'?  A lawn is a thin layer of confluent bacterial growth, usually created by mixing a relatively large number of cells (≥10^6) with liquid agar solution ('top agar' or 'soft agar' and pouring…

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Questions about CRISPR-mediated phage immunity

Thursday's post described the hypothesis that bacteria might use gene transfer agent particles to inoculate other cells in the population with fragments of phage DNA, and outlined an experiment to test this.  Now I'm realizing that I need to know a lot more about the kind of immunity I should expect to see if this GTA-as-vaccine hypothesis is correct.Simplistic outline of the experiment:Infect GTA-producer strain of R. capsulatus with phage under conditions where the infection…

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Why GTA genes can’t be maintained by ‘selfish’ transmission

Below is the line of reasoning showing that the genes responsible for producing GTA particles cannot maintain themselves or spread into new populations by GTA-mediated transfer of themselves into new cells.  I initially worked this out with a rigorous set of mathematical equations, but then realized that the problem was so glaringly obvious that math isn't needed.The main GTA gene cluster is too big to fit inside a single GTA particle, so GTA particles can't…

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Why GTA genes can’t be maintained by ‘selfish’ transmission

Below is the line of reasoning showing that the genes responsible for producing GTA particles cannot maintain themselves or spread into new populations by GTA-mediated transfer of themselves into new cells.  I initially worked this out with a rigorous set of mathematical equations, but then realized that the problem was so glaringly obvious that math isn't needed.The main GTA gene cluster is too big to fit inside a single GTA particle, so GTA particles can't…

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Might GTA be a vaccination system for infecting phages?

My work at Dartmouth (to be described in upcoming posts) showed conclusively that genes encoding Gene Transfer Agents (such as the GTA system of Rhodobacter capsulatus) cannot be maintained by 'selfish' transfer of either whole GTA gene clusters or single GTA genes into GA- recipients.  Neither can the GTA genes be maintained by general recombination benefits that can arise when fragments of chromosomal DNA are transferred into new cells.  So, although 'gene transfer agent' does…

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Thinking about Gene Transfer Agent

I'm at Dartmouth for three months, working with Olga Zhaxybayeva's group to improve our evolutionary understanding of Gene Transfer Agent.  I'm writing an R-script simulation of the genetic exchange it causes (finally learning R), but my control runs with epistasis don't give the expected results.  So I'm writing this post and creating a Powerpoint deck to clarify my thinking.First, what's Gene Transfer Agent?  A number of different kinds of bacteria produce 'transducing particles' called Gene…

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