Proposal priorities

I've gone through our 2007 CIHR proposal, incorporating my polishing of the text and notes about things the reviewers liked or didn't like. I also added notes about what we could reasonably accomplish before Sept. 15; here is an updated version of the ideas I posted a few weeks ago.

Specific Aims:

1. "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." I've been getting info about designing and ordering the degenerate oligos. The post-doc had already set up a spreadsheet that does the calculations I wanted to think about, and he and I agreed that we should start with 12% degeneracy rather than 9%. This will reduce the fraction of oligos that are strongly preferred, giving more sensitivity for detecting weaker effects. I've had replies from two custom-oligo companies, and the good news is that our degenerate oligo pool will be easier to get and much cheaper than I had expected. For the proposal we'll only need to do some conventional sequencing, and as our USSs will already be in plasmids we think we'll just sequence each plasmid insert separately. This will be wasteful but not very expensive if we do the DNA reactions and cleanups ourselves, and probably cheaper when we consider the time/money we'll save by not having to troubleshoot a more 'efficient' sequencing strategy.
   
2. "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." My physicist collaborator is keen to have me back to get this working. The only think I could aim to get done before September 15 is to attach some chromosomal DNA to the styrene beads and show that Bacillus subtilis will bind to it and pull on it in the tweezers apparatus, and that H. influenzae doesn't stick nonspecifically to beads with no DNA on them (a concern raised by a reviewer). The biotin-linked chromosomal DNA I'll use for this will also be used in other preliminary experiments.
   
3. "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." Our 1 micron paramagnetic streptavidin beads are on their way, and I'll use these to check for non-specific binding of cells to beads and for specific binding of competent cells to beads with DNA on them (using the same DNA prepared above). I found the source of the 50 micron beads and will order them tomorrow; maybe I can use them in the same way.
   
4. "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." I'm going to try the nicking protocol tomorrow.
   
5. "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." We'll have the DNA on the big magnetic beads (see 3 above) and can use this to try out the formaldehyde cross-linking. It would be good to show that we can distinguish between non-specific proteins or peptides (independent of both DNA and competence) and peptides cross-linked only when cells are competent and the beads have DNA on them. One of the reviewers really liked this part, but thought we should be more ambitions and propose more diverse approaches to this problem, including in vitro cross-linking to purified secretin.
   
6. "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." It would be good to have results of some complementation experiments with single gene plasmids, as the whole-operon plasmids seemed to cause growth problems.
   

The RA will be back on Monday, and she, I and the post-doc will sit down together to work out who can try to do what. Then I'll start thinking about integating these experiments and the post-doc's experiments into the NIH proposal.