Proposals plan

Last week I went down to Bellingham to collect pearls of wisdom from a friend and colleague who has lots of experience with NIH. I came away with lots of good ideas for strengthening our NIH proposal, but the most important was the decision to delay submission to the next deadline, in February. This will not only let us generate more preliminary data but will let me get the US-variation manuscript submitted. This submission is important because I now realize that its contents will play an important role in the proposal.

So for the next two weeks I'll only have to think about the CIHR proposal. It's due Sept. 15, but I'll need to click the submit button Sept. 13 (Sunday), because I'll be traveling on the 14th and 15th. (The paperwork (electronic and real) needs to be in a few days before that.)

Although the reviewers thought that the previous version of the proposal was well written, I now see that the overall structure was weak. In particular, the Introduction (a short section, only 3 paragraphs) introduced competence but failed to introduce the problem/s I want to address, the hypothesis/hypotheses I was testing, or the approach I will take to test the hypothesis/hypotheses. That was because I hadn't done a good enough job of spelling these out even to myself. So here's a new attempt:

The phenomenon: Bacteria actively transport DNA molecules into their cells, across their outer and inner membranes.

The specific problems this proposal addresses: How does H. influenzae transport DNA across its outer membrane: (a) How is this uptake initiated? (b) How are long fragments pulled into the periplasm? (c) Why is uptake so strongly biased towards specific sequences?

The hypotheses: (1) Initiation and progression of DNA uptake are mechanistically distinct processes. (2) Uptake sequences are favoured because they facilitate the DNA deformation needed to initiate uptake. (3) Progression of uptake uses an ATP-driven ratchet driven by assembly and disassembly of type four pseudopili (this hypothesis may well be wrong, but that's OK).

The Specific Aims: (1) We will fully characterize the uptake specificity, using both synthetic sequences and the full H. influenzae genome. (2) We will identify the proteins that contact DNA during uptake and those responsible for the sequence specificity. (3) We will test whether the uptake sequence motif polarizes uptake and whether it promotes DNA kinking. (4) We will characterize the forces acting on DNA during binding and uptake.

I'm going to paste this summary on the first page of my draft proposal, so I incorporate it into the Introduction and use the Background to establish the problems and build to the hypotheses.