H. influenzae is reported to efficiently take up closed circular DNA as well as linear DNA. The DNA can be re-isolated intact after the cells have been treated with DNase I, so it is thought to be in the periplasm. I've based much of my thinking about the mechanism of DNA uptake on this result, but I've never tested it for myself. Now's the time, because the ideas in the DNA uptake proposal depend on this being true.
How to do it? How was the original experiment done? (First step is to find the paper...) Would they have used radioactively labeled DNA, and if so how did they label it without compromising its supercoiled state? (Prep the plasmid from E. coli grown in the presence of massive amounts of 32-P? Yikes!) Or maybe they did a Southern blot to detect it? Find the paper...
Basic plan: Incubate competent cells (wild type or rec-2 mutant) with supercoiled plasmid containing a USS - pUSS1 isolated from E. coli using a kit should be fine, except for the labeling question. Treat cells with DNase I and wash them thoroughly to get rid of external DNA. Lyse cells and prep DNA using a plasmid-prep kit. Run in a gel. If the recovery is really high I might be able to see the unlabeled plasmid, especially if I use one of those ethidium alternatives that's much more sensitive. How high could recovery be? Say I have 10^9 cells, each takes up 10 plasmid molecules. That's only 10^10 plasmids; if they're 2kb long that's 2x10^10kb of DNA, which is about 0.02 micrograms. 20 ng can easily be seen in a gel, especially with a fancy dye, but I'd be out of luck if uptake is less or recovery is poor (for example, if the DNA is nicked the kit won't work).
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You could detect your plasmid by PCR, so long as control bacteria gave no signal. You might not need to do a full prep to get clean enough DNA for PCR, either.
ReplyDeleteQuantitative PCR, OK (traces of plasmid would certainly be present in the no-uptake control). But I also need to detect its topological state (is it still supercoiled), so a gel would be much better. Need to find that paper...
ReplyDeleteWhy the interest in supercoiling? Cells take up linear DNA, so recognition of the USS probably isn't dependent on a specific topological state (except that supercoiling might interfere with protein-DNA binding if the machinery is evolved to bind linear DNA fragments). Do you think that supercoiled and linear DNA is transported better across the cell envelope than is relaxed circular DNA? If supercoiling isn't critical, could you: digest plasmid, label with P32 end-filling, then circularize with ligase and use an exonuclease (like Lambda exonuclease) to remove any non-circularized DNA.
ReplyDelete@Andrew alias Tim: Supercoiling is important only because if it is retained when plasmids are transported into the periplasm we can be sure that the DNA wasn't nicked during uptake and later resealed. That's pretty unlikely so maybe labeling as linear DNA and then sealing the ends is a good plan.
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