Basically it's a lovely proposal with one massive flaw. The experiments are a fine mix of safe and risky, and the proposal is beautifully written and illustrated. BUT, there's no preliminary data to show that the experiments we propose are likely to work. (The reviewers also grumbled that I was being sneaky in submitting proposals to two different committees in the same competition. Such greed is, of course, un-Canadian, but that won't be an issue this time.)
So now it's time to think more strategically (i.e. about strategy) than I usually do. Which of the 6 lines of investigation I propose are easiest to generate preliminary data for? Which are the most severely weakened by their lack of preliminary data?
Here are the Specific Aims from that proposal, with brief notes about preliminary data in italics:
- "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 about to start doing preliminary experiments for this and related work, so we should at least be able to show that we can reisolate DNA that has been specifically bound by competent cells.
- "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). We have all the components ready, and I'm keen to work on this myself.
- "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." We've tried this unsuccessfully in the past with much bigger beads. The magnetic beads may also be valuable for the post-doc's DNA-reisolation work - I wonder what form preliminary data should take.
- "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." We should at least try this cyclization assay - it shouldn't be a big deal.
- "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...
- "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.