The other project I'm taking on is the controls and preparation for our laser tweezers analysis of DNA uptake. Our previous work (really the physics grad student's work) was trying to get cells to attach to and take up a specific bead-attached DNA fragment with a single USS. If this had worked we hoped it would let us study how DNA uptake depends on the orientation of the USS. We now realize that this was too ambitious an initial goal.
Our new experimental goals are to study the forces that act on the DNA during uptake, in wildtype cells and in cells with mutations in specific uptake proteins. This doesn't require use of a defined DNA molecule with only one USS, so we can change the setup to optimize our chances of success.
We also belatedly realized that we can use Bacillus subtilis as a positive control. Laser tweezers have already been used to study forces generated during B. subtilis DNA uptake. (Because the B. subtilis uptake mechanism is quite different than that of H. influenzae this serves as proof-of-concept for our experiments but doesn't make them redundant.) B. subtilis cells are larger and more robust than H. influenzae cells, and their DNA uptake does not depend on a particular sequence. Because the conditions for B. subtilis tweezer assays have already been worked out, we will make sure we can demonstrate uptake by it before moving on to uptake by H. influenzae.
The new plan is to first make sure we have lots of DNA fragments attached to each polystyrene bead, before trying to detect uptake by either B. subtilis or H. influenzae. My first step is to get a preparation of randomly broken H. influenzae DNA that consists mainly of fragments 50-100kb long, and then to attach these fragments to the beads by sticking biotin on the ends of the DNA, and using beads pre-coated with streptavidin, which binds tightly to biotin.
I've decided on 50-100kb for several reasons. First, it's hard to work with fragments longer than this, because longer fragments break unless they're handled very gently. Second, it's easy to get fragments about this size simply by not being gentle with the DNA prep. Third, this will let me attach a LOT of DNA to each bead. Fourth, the individual fragments will be substantially longer than the cells and the beads: the beads are 1 micrometer in diameter, the cells are 1-3 micrometers long, and a 60kb DNA fragment is about 20 micrometers. But they aren't so long that they extend a long way away from the bead they're attached to.
I'll use DNA from strain MAP7, partly because we already have lots of it (made by one of the post-docs) and partly because it carries antibiotic-resistance alleles that we can use to detect whether cells have taken it up. This will be important in checking whether the beads really do have DNA attached to them.
The DNA in the prep we have may already be broken into appropriately-sized fragments. I can check for small pieces by running this DNA in a normal agarose gel; fragments bigger than about 25kb will all jam up at the top of the gel, and only smaller fragments will spread out in the gel. A better check for DNA size will be to run the DNA in a pulsed-field agarose gel, whose rapidly reversing electrical current allows even very big fragments to spread out in the gel. Luckily our neighbours in the lab have a new apparatus to run these gels. (We have a 15-year old system which I suspect no longer works.)
If this analysis says the DNA fragments are too small, I'll make a fresh prep, handling it more gently. If the analysis shows that the DNA fragments are too big, I'll just rough up the DNA prep by whirling it in the vortex mixer, or by forcing it through a narrow syringe needle.
The next step will be adding the biotin to the ends....
(Hmm, the spell-checker thinks that tweezers must be plural; it doesn't approve of "laser tweezer analysis".)
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