Monday, August 10, 2009

Back to the grind

Whew! Getting that albatross (= grant application) off my neck feels good, but now I need to formulate a plan for the next several weeks of lab work. What are my priorities? There’s quite a lot of things I could be doing, so I might as well make a list...

(A) Generate purified DNA from 86-028NP, PittEE, and PittGG donor DNA: Should be pretty easy. I tried this a couple times in the past couple weeks, but had a few problems:
  1. Got a goopy mess. I don’t know if it was my lysis or something about my extractions, but when I would try to pull the aqueous layers off of my phenol/chloroform extractions, this goopy junk at the interface kept interfering with my ability to complete the preps. I will try try again, but this next time I will do a few things differently: (i) start with fewer cells per volume, (ii) do a straight phenol extraction first, (iii) let lysis proceed longer.
  2. PittEE didn’t grow. I’d gone to the lab stocks, streaked for single colonies, grown up overnight cultures from single colonies, and stored them in glycerol in the -80. But when I returned to PittEE frozen stocks, they don’t grow up! Weird. Maybe PittEE is particularly bad at surviving outside log-phase, so I had merely frozen down a pile of dead cells. I’ll have to return to the lab stocks and restreak for single colonies.
  3. I never checked the putative nalidixic acid resistant phenotype of 86-028NP. I really hope that marker will work to select for transformants from 86-028NP to KW20, as this would really help several steps of the project. In our strain database, 86-028NP is listed as NalR, but this might only be a clinically relevant phenotype. I need to check it on plates.

(B) Check the uptake and transformation phenotypes of WT, rec-2, rec-1, and pilB: I’ve already gone through this exercise with wt and rec-2, but as a negative control, pilB mutants should neither take up DNA nor be transformed by it, and for my translocation experiments, rec-1 should take up DNA normally but fail to transform. I have all these strains frozen down as M-IV competent cells, so I just need to do the experiments to verify that my mutants have the expected phenotypes. The rec-1 mutant in particular is important, since I plan to use it for cytosolic DNA purifications, but it could be somewhat leaky, something I need to know. (The only rec-1 mutant available is the original isolated mutant from back in 1972. It’s older than me!

(C) Quantify uptake and transformation for different-sized donor DNA: A coupe weeks ago, I used a “Bioruptor” to sonicate MAP7 DNA to different size distributions, and I tested the transformation rate of antibiotic resistance into competent cells of some of these different sized DNA. I also want to check how well they’re taken up. Once I’ve done this, I’ll report back the full set of results. The expectation is that while even small fragments will be taken up well, only large fragments will transform well. There’s also an interesting expected relationship between the amount of uptake expected and the size distribution of the DNA. If the DNA fragments are quite small, then only a fraction are expected to contain uptake signals, but many fragments containing such signals will be taken up. On the other side, large DNA fragments will mostly all contain uptake signals, but only a few molecules will be taken up, but these will be larger... Hmmm....

(D) Design the degenerate USS construct. This will require its own post, but suffice it to say, we have a pretty good plan on how to design this construct so that we will be able to directly sequence the degenerate USS motif by short single-end Illumina sequencing without any processing steps upfront.

(E) Transformation experiment: If indeed 86-028NP can grow on nalidixic acid plates, then I will just go ahead and do half of the experimental-side of one of my specific aims. All I need to do is transform KW20 competent cells (which I have) with DNA taken from 86-028NP (which I’ll make), select for colonies that are NalR, pick several of them, grow them up, and extract their DNA. This would provide up with plenty of material to produce sequencing libraries and then send to sequencing. Then we can really get this project off the ground.

(F) Find somewhere that will do sequencing with a short turn-around: The local Illumina sequencers (at UBC’s Genome Center) offer what looks like excellent sequencing services for a reasonable cost, but their turn-around time is a bit too slow for us to hope to have any preliminary data for Rosie’s grant applications. If we want to get anything done more quickly, we will have to find someone else to get started.

(G) Design and produce a large DNA fragment from the 86-028NP genome to use for developing a cytosolic ssDNA prep: Since the most difficult experimental part of our plans is likely to be purifying (and cloning) ssDNA from the cytosol, I’d like to start with a defined construct I can use for purification experiments. I think that a good choice would be a large clone from 86-028NP, because this might be useful for other experiments as well. I will want something at least several kb on a plasmid. I will need to think about whether or not it matters what this fragment contains. As a first guess, I would want it to contain the putative NalR allele and several kb of flank.

What else should I be doing?

1 comment:

  1. Why not just use linearized pUSS1 for the cytosolic reisolation tests? How 'large' does it need to be?

    I would use the simplest already-available DNA fragment for these tests. If you need a 86-028NP chromosomal fragment for other tests, optimize it for those tests.

    Can we (you) think up a clever test molecule for the cytosolic reisolation tests? It probably should share no sequences with the KW20 chromosome, so you can be sure that what you've isolated is your input DNA.