The other day I tested for phage recombination, using my new mutant phage lysates and incubators. As expected, I saw lots of recombination in competent cells (I didn't test non-competent cells). Today I tested the postdoc's hypothesis that recombination occurs by recombination between the DNA of infecting phage and strands of phage DNA that was free in the lysate and was brought into the cytoplasm by the competence machinery.
I used two lysates of phages carrying different temperature-sensitive mutations. An aliquot of each lysate was incubated with DNase I, which should destroy the free DNA and prevent recombination if this hypothesis is correct. A second aliquot of each lysate was incubated with Proteinase K, a broad-specificity protease that should degrade enough of the phage protein that they are unable to infect the cells. This treatment should not harm the DNA, and if the hypothesis is correct, it should reduce the infectivity of the phage but not reduce recombination.
I infected competent cells with the untreated lysate of each phage in combination with each of the treated and untreated lysates of the other phage, and assayed both the infectious centers and the final lysates for recombinants by plating at both 33°C and 41 °C. Tomorrow I should have the answer.
(I also treated the last four of our competence mutants with the competence-inducing ritual, transformed them with novobiocin-resistant DNA, and froze aliquots for later recombination and uptake assays.)
21 hours ago in Variety of Life