I've started polishing my not-quite-good-enough CIHR proposal for what's called the 'Transitional Open Operating Grant Program' competition. This is the last-chance-under-the-old-system competition; any future proposals will be evaluated under the new system, which doesn't have much use for pure science or small labs. Proposals are due March 2 2015.Before then I'd like to do some preliminary experimental work on one of the studies I'm proposing. I expect that the H. influenzae DNA available to H. influenzae cells in the host environment will still have bound to it many of the normal chromosomal proteins (HU, H-NS. Fis), and might even retain aspects of the normal nucleiod structure. I want to find out how this affects the ability of the DNA to be taken up by competent cells, particularly whether specific sequences or segments are affected more than others.
My poorly thought-out plan is to lyse donor cells carrying one or more antibiotic resistance alleles in a way that doesn't disrupt bindings of proteins to DNA (so not with 1% SDS), and then mix this lysate with competent sensitive cells.
Big problems I forsee:
1. How to lyse the donor cells without disrupting the nucleoid proteins?
My original plan was to use the H. influenzae phage HP1.
I could probably instead lyse the cells with lysozyme and a small amount of a surfactant that disrupts membranes but not proteins.
2. How to lyse the donor cells without killing the recipient cells?
If the recipient cells are lysogenic for this phage (I have such a strain in the freezer), then they will be resistant to the free phage in the lysate. An undiluted lysate has an enormous number of phage (>10^10 per ml), but I'll dilute the lysate to a chromosomal DNA concentration of about 1 µg/ml, which would be saturating for uptake if the DNA had been purified.
(Hmm, what will be the chromosomal DNA concentration of a lysate? Say 3x10^9 cells/ml, most of them lyse, 1,830 kb of DNA/cell, 10^-12 µg of DNA/kb... That's only about 4 µg of chromosomal DNA per ml.)I can dilute the DNA way more than this, because transformation has such a wide sensitivity range. I could also make it difficult for the phage to infect by eliminating Mg, but that might also hinder DNA uptake (the normal competence medium is 10 mM Mg). If I had antibodies to the phage I could block infection that way (we used to do this with lambda), but I don't.
If I lysed the cells with surfactant, I could then easily dilute the lysate to reduce the concentration of the surfactant to a concentration that wouldn't harm the recipient cells (10-fold or 100-fold wouldn't be a problem).
3. How to kill off or remove all the donor cells that aren't lysed, without disrupting the nucleoid proteins?
I don't know if it would be possible to pellet the cells without pelleting the nucleoids, especially since the nucleoids and DNA may be still attached to the cell wall. Or to filter out the cells without removing or disrupting the nucleoids.
I don't really need intact nucleoids, but I'd like to still have the proteins bound to much of the DNA.
How can I check the state of the DNA? I hope I wouldn't need to use electron microscopy. Maybe I could use a simple very-low concentration agarose gel? Like an Eckhart gel, but interested in the DNA+gunk, not the megaplasmid?
4. How to kill off or remove all the donor cells that aren't lysed, without killing the recipient cells?
If the recipient cells are already resistant to an antibiotic that the donor cells are sensitive to, I can include this antibiotic in my selective plates.
Or maybe I could kill them by adding a bit of chloroform to the lysate, and then diluting or evaporating the chloroform before I add the lysate to the cells. Chloroform is normally used to sterilize phage lysates, but I don't know if it would affect the nucleoid proteins.