Field of Science

The proposal is finally taking shape

My research proposal finally is reading more like a narrative and less like a disjointed string of ideas.

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.
MALDI-TOF spectrometry needs a very expensive machine and a trained operator - luckily our Proteomics Service group has this. But I'll need to go talk to them to find out the limitations. One that I'm especially concerned about is the amount of cross-linked protein we can get. We're limited by how competent our cells will be and how many fragments our preps will be able to take up.

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.

1 comment:

  1. Came across this ref on reversible cross linking using formaldehyde

    ReplyDelete

Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="http://www.fieldofscience.com/">FoS</a> = FoS