What will I talk about? I wish I'd been doing some experiments. What did I talk about last time? I don't have my notes here, but I think it was the US variation manuscript - I think I went through the figures. Since then I've mostly been working on the NIH proposal, so I guess that's the best thing to talk about.
The Specific Aims keep morphing. Here's what I thought was the stable version:
Aim 1. Characterize the recombinome by sequencing DNA from millions of pooled transformants.
Aim 2. Characterize the biases of the steps that make up the recombinome, by sequencing:
a) millions of chromosomal DNA fragments and degenerate uptake sequences taken up by competent cells,
b) chromosomal DNA fragments taken into the the cytoplasm of competent cells,
c) chromosomal DNA recombined by a non-RecA recombinase,
d) DNA of millions of transformants of a strain unable to do mismatch repair.Aim 3. Map loci responsible for transformation differences between two strains.
But the post-doc's new data suggests high mutation rates in recombined sequences, and this may mean that we should put more emphasis on what he calls the transmission genetics. That is, we should first pin down the general properties of what actually gets recombined. How much DNA is typically replaced? In how many segments? What is their size distribution? Are tracts of donor DNA interrupted by recipient alleles? Do indels recombine cleanly, by homologous recombination in flanking sequences, or do we see non-homologous recombination at one end? What is the frequency of new mutations in recombined DNA (and in the rest of the genome)? This information is best obtained by sequencing the DNA of individual transformants, not a big pool.
This should probably become (part of?) our first Aim. Should it be an Aim in itself? Will we then have too many Aims?
I can see two different directions we could take this: I. We could make a big shift in direction, getting rid of all of Aim 2 and expanding Aim 3 to include more strains. That would give a much more streamlined proposal, one focused on the consequences of recombination and not the component steps. Or II. we could get rid of Aim 3, keeping the focus on the processes that give rise to recombinant genomes. Both are areas I think are really important. Direction I fits well with the work a previous post-doc did, characterizing the variation in DNA uptake and transformability of a wide range of H. influenzae strains. Direction II fits better with my desire to understand the role of uptake sequences, but this goal is really only addressed by Aim 2 a, and that's already included in another proposal, the one we submitted to CIHR in September.
In either case we should probably demote Aim 2 c to an optional goal, unless we can get the data showing that this alternate recombinase (lambda Red) does indeed work in H. influenzae. That would be a very cool result, but it's not central to this proposal.