Aim A. Map the H. influenzae recombinome. This is basically a higher resolution version of what we just proposed to Genome BC. We will propose to use next-gen sequencing to map the frequency of recombination at all the >40,000 SNPs and >300 indels/rearrangements between two H. influenzae strains, Rd and 86-028NP. These frequencies will be the net effects of biases at many levels of the transformation process.
Aim B. Characterize the components contributing to the recombinome of strain Rd.
- Uptake bias (across the outer membrane)
- Translocation biases (across the inner membrane)
- Cytoplasmic biases (nucleases and protection)
- Strand-exchange biases (RecA-dependence)
- Mismatch repair biases
To do this we will use various combinations of mutants and experimental manipulations to (i) eliminate other effects and (ii) generate DNA samples enriched for the effects of the component of interest. For example, we can get a DNA sample enriched for preferred uptake sequences (step 1) by giving rec2-mutant cells synthetic degenerate uptake sequences. We can see the effects of repair (step 5) by comparing the Aim A recombinome with one produced by mismatch-repair mutants. Maybe we can detect RecA's contribution (step 4) by repeating the analysis in rec1 cells expressing the lambda bet recombinase. We can propose lots of different analyses here, giving some high priority, tagging others with 'if time and resources permit', and leaving some as 'future directions'.
Aim C. Develop a genome-wide map of genes contributing to the transformability differences between these two strains.
I don't seem to have posted about this before, and the post-doc hasn't either (it's his idea). I think it deserves a separate post. I'll just say here that this will be presented partly as a proof-of-concept study for a method that can then be used to map genes contributing to any selectable trait.
Aims B and C both depend on first completing Aim A but they are independent of each other. They also share the lovely property of being open-ended, putting us in the best version of Hershey Heaven.