Ideas from lab meeting

At today's lab meeting we discussed the feasibility of the six Specific Aims from the 2007 CIHR proposal on DNA uptake, and considered what kinds of preliminary data we might generate. I'll discuss each separately here.

• "What is the H. influenzae uptake specificity? A pool of USSs that have been intensively but randomly mutagenized and then selected for the ability to be taken up by competent cells will be sequenced to fully specify the uptake bias."
  

The new-post-doc is beginning experiments to show that he can reisolate DNA that has been specifically bound by competent cells. He also suggested a better way to make the pool of randomly mutated USS. Rather than using the degenerate 30-bp USS oligos as primers with a mutagenesis kit, he suggests instead creating 50- to 60-bp oligos with 20 bp at the 3' end outside of the USS. He'll then use Klenow polymerase to fill in the second strand of these oligos, with a primer complementary to the 3' end, and clone these to make our diverse library of degenerate USSs. Sequencing of some of these will provide preliminary data that our input is correct.

• What forces act on DNA during uptake? Laser-tweezer analysis of USS-dependent uptake by wild type and mutant cells will reveal the forces acting on the DNA at both the outer and inner membranes.

This work was begun by a physics grad student from the lab with the tweezers (across town). I subsequently revised the plan to make things simpler, more efficient, and better controlled. I have a stock of the styrene beads somewhere (with streptavidin), and I've already checked that our standard MAP7 DNA has a suitable length distribution. So I need to put biotin ends on the DNA. I have biotinylated nucleotides but don't remember how I was going to attach these to the DNA ends. I could cut the DNA with a rare-cutting restriction enzyme (SmaI?) and then fill-in with the nucleotide (better read my old notes). SmaI sites are sufficiently rare that most molecules in the MAP7 DNA prep would have biotin only on one end. Then I need to bind the DNA to the beads. That's easy, but how do I check how much DNA is on the beads? Measure DNA concentration by Absorbance at 260? (Does styrene absorb UV?) Then I need to arrange with my physicist collaborator to try them out, using Bacillus subtilis first because its cells are nice and big and it doesn't care about the sequence of the DNA it takes up.

• Does the USS polarize the direction of uptake? Using magnetic beads to block uptake of either end of a small DNA fragment will show whether DNA uptake is symmetric around the asymmetric USS.

This time I need to lay out clearly how answering this question would help distinguish between different models of uptake. We tried this experiment unsuccessfully about 5 years ago with much bigger streptavidin-sepharose beads (we later realized that the beads were way bigger than the cells, and porous to boot). I need to do more careful thinking of how this would be done and what we might measure.

• Does the USS increase DNA flexibility? Cyclization of short USS-containing fragments will reveal whether the USS causes DNA to be intrinsically bent or flexible, and whether ethylation or nicking can replace parts of the USS.

The RA pointed out that a former post-doc tried unsuccessfully to get this cyclization assay working. The problems are all nicely recorded in her blog, as well as being documented in her lab book. Hmmm, what to do? I found a 2009 paper about DNA kinking that used nicking and 2 nt gaps as controls; they didn't see significant kinking with their nicked DNAs. I was going to test whether ethidium causes restriction enzymes to nick circles instead of cutting both strands, but the post-doc and RA tell me that we can now buy 'nickase' derivatives of restriction enzymes instead.

• Which proteins interact with incoming DNA? Cross-linking proteins to DNA tagged with magnetic beads, followed by HPLC-MS, will be used to isolate and identify proteins that directly contact DNA on the cell surface.

It would be good to at least show that the cross-linking works... The RA says that she and a former post-doc tried using formaldehyde to cross-link DNA and proteins and got nothing but gunk. So I think this will take a lot of optimization. Start by getting the magnetic beads on the DNA. Start by ordering some magnetic beads.

• Which proteins determine USS specificity? Heterologous complementation with homologs from the related Actinobacillus pleuropneumoniae (which recognizes a variant USS) will identify the proteins responsible for this specificity.

I made the plasmids for this work several years ago, but they were not well-tolerated by the host cells. I should pull them out and see if I can get some results.

Before any of this, I or the post-doc should carefully test whether H. influenzae can indeed take up closed circular DNA intact. I've been citing an old report of this for years, but it's critical to my model of DNA uptake so I really need to test it myself. I'll write a separate post about this.