One experiment that I'll be looking for advice on is to cross-link transforming DNA to the proteins that are taking it up, re-extract the DNA with its attached proteins, undo the cross-links, and identify the proteins.
- The DNA will be our USS-1 fragment, about 200bp with a perfect USS in the middle. It will have a biotin molecule on each end.
- The 'taking up' could be done by cells, but we'll get cleaner results if we can start with a preparation of cell membranes, or blebs ('transformasomes') or pili, i.e. material enriched for the uptake machinery but unable to translocate DNA across the inner membrane.
- The cross-linking will probably be done with the chemical formaldehyde, because these cross-links can easily be undone (though I don't know how).
- Re-extraction of the DNA plus any cross-linked proteins will use the biotin tags, by mixing everything with agarose beads covered with the biotin-binding protein streptavidin. We can easily then separate the beads, with their attached DNA with its cross-linked proteins, from everything that isn't cross-linked to the DNA.
- Then the cross-links will be undone and the DNA digested away with DNase I. (Maybe only one of these steps is needed?)
- Then the protein mixture will be examined by a mass spectrometry technique called MALDI-TOF, which separates the proteins by their size. MALDI-TOF gives very precise size measurements, and these may be sufficient to let us identify specific proteins.
- Identifications can be checked by repeating the cross-linking analysis with mutant cells lacking known proteins.
I also have the name of someone to consult with about the cross-linking. Given the limited number of uptake events we can have, we'll want the cross-linking to be as efficient as possible.