Field of Science

How best to test binding of competent cells to DNA on beads?

Now I have lots of biotinylated DNA, and a well-tested procedure for binding DNA fragments to streptavidin-coated styrene beads, I'm ready to test whether competent bacterial cells (B. subtilis or H. influenzae) will bind to the DNA on the beads.

How to do this isn't straightforward.  One problem is that the beads are about the same size and density as the cells (B. subtilis cells a bit bigger, H. influenzae cells a bit smaller), so once mixed they can't be easily separated.  That means I have no way to wash unbound beads away from cells, or unbound cells away from beads.  Another problem is that B. subtilis cells are known to cut DNA fragments as part of the uptake process, and in principle this might terminate uptake.  Though maybe not, as the cutting is part of the process that initiates uptake across the inner membrane.  H. influenzae cells don't cut DNA.

I could just mix competent cells and DNA-coated beads, both at low densities, on a microscope slide and watch for them sticking to each other.  Alternatively, we have some streptavidin-coated paramagnetic beads I could use - this would allow me to pull out the beads and see if cells had stuck to them.  But these 50 nm (super-tiny) beads, too small to see individually, so I'd have to plate them to see if there were cells there.  We might also have some micron-sized ones; I'll look around. 

OK, I found our 'starter kit' of 1 and 2 micron paramagnetic beads.  The only problem is, we were too cheap to pay the $150 for the starter version of the magic magnetic rack that holds microfuge tubes against magnets so the beads stick to the side and the liquid can be removed. So I tested various magnets from around the lab, and all of them pulled the rusty-brown beads to the side of the tube in a couple of minutes.  Doing this in a way that holds the tube steady so I can remove the liquid...not yet.

I'm away for a few days, but when I get back I may send an email out asking if anyone in the building has a Dynabeads rack I could borrow for a little while.

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