Lunch talk is done. Was rather terrible, in my own thoughts. Finishing it too close to the deadline, in combination with little practice rehearsing it & too much coffee throughout the morning means I stumbled (hard) a few more times than I would have liked. Half of my story was clearly a trainwreck (it’s been a messy project to date), while the latter one was pretty nicely formed (as it’s been pretty clean & straightforward so far). Oy vay.

I think I’m having some wine with (or for) dinner once I get home and get tonight’s exercises out of the way with. And maybe a full night of sleep, while I’m at it…

December 28 – Achieve. What’s the thing you most want to achieve next year? How do you imagine you’ll feel when you get it? Free? Happy? Complete? Blissful? Write that feeling down. Then, brainstorm 10 things you can do, or 10 new thoughts you can think, in order to experience that feeling today.


What do I want most? Get published three times in scholarly journals! Namely, in order that I envision the papers coming along, Molecular Microbiology, Journal of Biochemistry, and then finally Proceedings of the National Academy of Sciences.

How will I feel? Seriously? Is that a legitimate question here? I am going to feel fucking awesome!!!

Ten things I can do to get there? Oy vey…here comes the trouble.

  1. Figure out what alternative pathway hda seems to be mucking around with, either genetically or biochemically.
  2. Purify a bunch (more) Hda protein and demonstrate that it definitely impairs translesion synthesis DNA polymerases.
  3. Demonstrate that Hda is able to stabilize the replication fork and alter the ability for other accessory proteins to interact with the complex.
  4. Show biochemically that my mutant library is composed of both RIDA-competent and RIDA-incompetent mutants.
  5. Get a crystal structure of Hda, or a co-complex of Hda with the ß-subunit of DNA polymerase III.
  6. Selectively break interactions of Hda with other accessory proteins, and restore those interactions by compensatory mutations on said accessory proteins.
  7. Genetically demonstrate the ability to block and/or promote these interactions with mutants.
  8. Demonstrate the blatant redundancy of the initiation regulation pathways in cellular viability.
  9. Demonstrate that accessory functions of the initiation regulators were the evolutionary pressure for adoption as opposed to control of initiation.
  10. Make the grand corollary of what all this means in the greater scheme of breaking the microbial replication cycle and propose a new, unique pathway of antimicrobial targets.

Nobody said I couldn’t be dreaming when I thought all of this up. But it’s an entirely possible order if the experiments go in my favor…

This has been a week from hell. My stress load was through the roof, and I really thought I was going to crack and lose it a couple times throughout.

Especially Tuesday; it had turned out to be a 14-hour day whereupon I went home, had two beers, and then promptly slept.

Wednesday, I got some drinks and got to sleep semi-early that night.

Thursday, I left for work at 6:45am, went to Alternative Brews at 8pm for the Jets/Bills game in Toronto, went to Denny’s (under some mild peer pressure) after the game, and finally got home at nearly 1am. Less than enthused about the time at the bar1.

Friday, left for work again at 6:45am2 and took off semi-early so I could get a (dearly) needed haircut, deposit my paycheck, and then finally recover some. I really stood around bewildered for about five or ten minutes once I got home. I honestly can’t remember what I’ve done here all week besides sleep and eat breakfast. My dirty dishes for the whole week consisted of two plates and four bowls, plus some containers of food I had just emptied out ’cause they went bad. I’m not even sure what nights I ate dinner. Huh.

Committee meeting is this coming Wednesday. That’s why I’m stressing. It’s been a bit of a crappy year again, and I get ridiculous amounts of anxiety over the concern of having not achieved enough in my research. Talking with my advisor yesterday morning, he said his goal for me at this point is to get me a solid paper and a good post-doc lined up. It was disheartening to hear that. I really still had some hope/ambition for pumping out at least two if not four papers. I can get the second one if I can for-god’s-sake purify my damn protein, but without that I’ll be resorting to primarily genetics to sort out my protein and all of its suppressor mutations. Less than ideal for what I had hoped.

Kelly made me want to watch all the Underworld series again. I’m out of the know for the first two; it has been a while since I’ve seen them. I think those will be interspersing my desperate attempts to finish my committee meeting presentation this weekend. That, and helping myself to more stuffing and pumpkin pie. I have approximately 2/3 of a casserole dish of stuffing and an entire pumpkin pie left to consume. In the succinct words of Liz Lemon…

Blergh!


1The bar was also a licensed cigar bar as well, so the place reeked of smoke and active smokers. I really hate (on many different levels) smelling like smoke when I go home.
2There was a Lake Effect Snow Warning for the metro area, and I’ll be damned if I’m going to drive in fresh, slick, wet snow with all the other rush-hour idiots, especially if my tires are on the balding side of wear.

Labwork has been frustrating lately. My favorite protein (MFP) has been less than cooperative over the years. First, it was a royal pain in the butt to purify to any reasonable degree. At best, I had to settle for a co-purified complex that was for the most part functional, but later to be demonstrated invalid. Last winter just after my last committee meeting, the original lab working on MFP published data stating that they have been working with a protein with an incorrect translation start site. In more lamen’s laymen’s terms, it meant the protein I had originally purified was wrong; the protein was about 15 amino acids too long. So since then, I’ve been trying to get this protein re-established and purified using their new protocol so I can redo all my old biochemistry, and finally move forward with the new biochemistry I had proposed to do. Well, it’s been nearly a year, and now I think I finally have some hope of purifying it.

    Issues that I’ve had to address include:

  • Poor expression (to the point I can barely see my protein above background cellular proteins)
  • Inability of affinity tag to stick to affinity resin (or any resin for that matter, ion exchange included)
  • Poor plasmid maintenance after an overnight culture (and have nothing left to grow the next day)

Since then, I’ve recently introduced a new selection marker that’s far less likely to be ignored that now seems to be giving me appreciable yields of expression in a rich, defined media (instead of our typical undefined media). So right now, I’m ready to crack open all my cells and see just how soluble my protein is. If it’s well-behaved in solution, then it’s on to the FPLC and see what I can get this sucker to stick to!

I also need to submit my construct for DNA sequencing; I really want to make sure that MFP’s coding sequence is pristine. No gaffes this time! I have to sequence a bunch of my selected mutants that have passed muster so far. Up to nearly twenty candidates and I need to get some sequence back to see what kind of mutations (if any) I am getting in MFP.

Additionally, since the “correct” translational start site is supposedly published now (nearly a decade since the lab originally identified the protein in the organismal genome), we’re not entirely convinced ourselves to run headlong into this and just trust it. I did get kind of screwed by trusting the original research in the first place, no? So, I had vocalized my concerns with this being an accurate piece of data, being as how they minimal upstream sequence to incorporate any endogenous promoter sequence. Well, that came back to bite me in the rear today; my advisor would like me to affinity tag my protein with an endogenous promoter sequence and get this guy purified and sequenced by Edman degradation to verify the start site. I don’t mind cloning, but I do sort of loathe designing primers for PCR. So that’s my new side-project to verify everything is kosher, so to speak.

The genetic selection library is coming along nicely, but the undergraduate who is working on the screening is a different story. I’ll save that for another night, so I don’t unnecessarily rip on her. She does a good job, don’t get me wrong! She just tends to miss some crucial steps on the way…or a lot…yeeeeeeeah.

I’ve been a bit busy lately dealing with a lot of real life stuff. This week has kind of been a symposia week, even though there’s only two that I’m attending. Just nailed one out of the way today, with one more to deal with on Friday. I’ll be presenting my own research then, so I really wanted to make sure that I had a decent presentation for it. I really don’t feel it’s as comfortable as I would like, but I don’t think I have much of a choice considering how my work has progressed lately. The ChIP isn’t moving along as nicely as I would like, but that’s another story for after I’m done stressing over symposia and talks and such.

For those that actually peruse this blog, look for a post in a day or two once I’m free from extracurricular obligations.