Switching gears...

I spent time today scanning NCBI for start and stop codons and got an email from my advisor about how we would discuss whether I should put my protein in a GFP or SUMO plasmid and what restriction sites would work tomorrow. I really am going into biochem land.

thesis

So I've got a thesis topic for my undergrad thesis. And a thesis meeting early tomorrow morning with my two advisors. I'm pretty psyched about it. I'm going to be co-advised by one of my favorite profs at Reed and someone who works in the area of natural product biosynthesis at Portland State University.

The project is going to be to clone and express two enzymes responsible for an interesting transformation in an antibiotic biosynthetic pathway. If I manage to get them pure I'm going to assay them with the appropriate analogs of the natural product and attempt to characterize them. One of them is interesting because it shows no sequence homology to the class of enzymes you would expect it to be in. It's required for the biosynthesis in knockout studies at the genetic level, though so it will be interesting to see what the deal is there (if I get that far).

So basically messing around with cloning and expression for nine months. Protein biochemistry is my one big gap right now in my lab training. I've got basic molecular DNA & RNA techniques down, I've done some cell bio stuff like ELISAs and Westerns and histology and various biochemical assays and shit, and I know how to do synthetic organic chemistry. But I haven't worked with cloning and expression and protein purification and all that stuff that makes you a real biochemist.

I hear protein work is boring as shit and frustrating as hell. But running silica columns is also boring as shit and can be frustrating as hell. So I guess I will finally have a true opinion of which is more boring: cloning or silica columns. And I can tell my chemist and biologist friends to STFU because I know all of boring, thank you very much, in all of its flavors.

Anyway, my general philosophy on this matter is that all fields of science has its boring and you just choose your boring and go with it. I can get through boring--the project just needs to be interesting. To quote the aforementioned Reed professor "Experiments suck. Until they don't."

lab notebooks

So here's something I've been thinking a lot about lately: lab notebooks.

Lab notebooks are something that I have a bit of a love/hate relationship with. I understand the value of a good lab notebook, and I try to write down everything that I do in it. I also find updating my lab notebook to be a rather tedious and annoying process. For the most part, my lab notebooks are reasonable, but the more complicated a project is and the more techniques I use, the more difficult I find keeping an accurate record to be, and my last lab notebook from this summer, while it contains everything that I did in it could be...uh...slightly better organized. When I was working on a project that was just organic synthesis, it was much easier to keep a good lab notebook.

But I guess, the strangest thing to me about lab notebooks is the fact that when you're done with a project, it stays in the lab with your boss. Of course it does; it's not your property. The record needs to be kept in case someone else needs to follow it. But it is a little strange to have something that's recorded in your handwriting, in your style, in a way that is comprehensible to you just gone, in someone else's hands forever. It's like parting with my diary, my record of my life for the past two months. It's strange.

Fuckity fuck fuck fuck

So I'm doing this experiment where I'm trying to get a bioengineered polyketide pathway to incorporate weird starter units. I did a small scale feeding experiment and got a promising looking result in terms of retention time, MS-2, and UV spectra. So then I did a 20L fermentation with hopes of eventually isolating it and getting an NMR.

First I did a huge ass silica column to get a crude seperation of the ethyl acetate extract. Then I did a huge ass sephedex column to get a better seperation. Then I did preparative HPLC to get 2.6 mg of something. I took a proton NMR. It was garbage. There were just too many compounds with similar retention times in there to seperate it, and the proton NMR was entirely too dirty to be at all informative. Basically, I couldn't see a lot of the diagnostic peaks at all, and a bit disheartening. BUT! It could have been that it was just too dirty. So on to further purification!

So then I purified my 2.6 mg on the analytical scale HPLC that has a really intensely good column with really great seperation. I fussed with the method with my supervisor for several runs and eventually got 2 peaks that had UV spectras consistent with my compound of interest (with this really fancy-pants analytical column, there were about nine peaks that showed up as one peak on the LC-MS spectra and maybe 4-5 peaks on the prep HPLC column, it was intense). I collected both peaks and started to evaporate them down on the rotovap using a vial adaptor that you can attach to the bump trap. One of my fractions was fine--I can put it on the vaccuum pump overnight, see if there's enough compound for an NMR (for the 500 MHz, you can get away with a little under a mg and still get spectra, so maybe if I isolate 0.7-0.8 mg of it I have a hope of doing the whole proton, 13C, COSY, HMBC, HMQC, etc. thing to really confirm the structure) and if not at least LC-MS it to see if it is there. There are more fractions from the spephedex column that one could purify--ones that actually had better seperation than the one I did attempt to purify (long story involving leaving in less than a week and the time it takes to get LC-MS samples and access to the prep HPLC in a large lab where a lot of people need the equipment)--but I simply don't have time to do it.

But the other fraction...well, what happened was a disaster. The vial adaptor LEAKED AND THE WHOLE VIAL AND BUMP TRAP FILLED WITH WATER FROM THE ROTOVAP BATH! FML!

So now I rinsed the vial and bump trap with MeOH, and am re-evaporating it and putting it on the freeze dryer overnight, hoping to god that a) something comes out, goddamn it, and b) it's not my compound of interest anyway (it was the less promising of the two peaks..but who really knows).

Seriously, though. FML.

I am going to have an honest appraisal of my scientific strengths and weaknesses. Let’s start with the negative:

• I have trouble with school. I’m not an abysmal student, but I’m not an exceptional one either. I have trouble with exams, and you’ve probably seen this whine before. For whatever reason, I have difficulty presenting information on a page that is demonstrative of my conceptual understanding of material—and all of my professors have commented on this. My current boss, actually, who is a brilliant PI, claims to have had similar problems as a student. He is extremely creative, has an encyclopedic knowledge of his field, is able to pull the right people together to manage a productive lab dynamic, is able to follow over 30 people’s projects (many of whom have multiple projects), and is able to publish in great journals in part due to his great sense of presentation and his ability to spin results into narrative stories. He tells us that he wanted to study biochemistry in university but his grades were too bad so he had to study chemistry instead (he’s German, so there you have to choose your field of study before you start), and that for whatever reason he just didn’t fit in the mold that school expects from you. My professors keep telling me that this will really only be a problem that will persist through my first two years of graduate school, and that it’s unfortunate, but wouldn’t it be worse if it were the opposite—I flew through my coursework and then I crashed and burned in a research project? I suppose that’s true, but it also makes getting into a decent program more of a crapshoot.
• I’m a little bit messy and spacey and I have difficulty with attention to detail. I’m just not a meticulous person by nature, and I tend to do spacey shit like letting something go on the rotovap for ten minutes before I realize that I forgot to shut the vacuum every now and then. In written work, I have difficulty with catching typos and small but stupid chemical errors like dropped carbons. Usually, at least in lab, when it’s really important I can focus, but sometimes…sometimes shit happens.
• I have a really shitty math background. Math has never been my strong suite, and I’m in a program that let me get away with taking very little math due to the way math is taught at our school (i.e. for mathematics students, we don’t have applied math classes for science students since it’s not the liberal arts “way”). Of course, I needed to get through physics and physical chemistry, which means I have some basic competence with algebra and calculus, but I imagine that if I ever need to take stat mech or biophysics or biophysical chemistry or anything like that in grad school, it’s going to be a fight. I just tend to look at scientific concepts in more pictorial terms.
• Adding to the "messy and inattentive to details" bit, at times I will cut corners that I maybe shouldn't cut when I'm under a time crunch. My lab notebook, for example, gets progressively worse the more I work on a project. Again, I think this is normal.
• I really can't handle extremely aggressive people. A lot of the cutthroat competitive behavior I find pretty distasteful and I'm generally pretty intolerant of that kind of BS. It strikes me that there are a lot of these type of people in science--especially science at a high level--and I need to learn how to deal with them.
  

Okay, the positive:

• I’m always praised on my creativity and the degree of sophistication that I have in putting together scientific concepts, and this has come from professors, my current boss, and the PhD students that I work with currently.
• I’m always praised on my ability to process the concepts and link them up to what I am physically doing in lab. One of my supervisors told me that I have such a high understanding of the theory that it’s often difficult for her to remember how much help I need with the little lab things that only come with experience, one example being how much trouble I had figuring out the right way to set up my first sephedex column. I had done many silica columns, but there are a few tricks that you need to do with sephedex columns that I just didn’t know. But she says that there are many undergraduates who come in and they don’t really have the big picture of what they are doing and why they are doing it.
• I’m very good at processing the literature. I know how to find papers and how to mine them for information. I also can competently read organic chemistry (synthetic, mechanistic, and natural products), biochemistry, and molecular biology papers. I’ve also been told that I know how to write about/talk about/communicate science in a story after reading papers/seeing talks/working on a project. At least profs tell me positive things when I turn in term papers.
• Sort of combining all of the above points together, I noticed that people tend to give me more autonomy in lab after I chat with them about science a bit. This is kind of strange, because just because I have the conceptual framework in my head doesn’t mean I need any less help getting the wet work to work. But I think there’s a psychological component with people where they are more likely to treat you like a colleague when your ideas are in place. That comment isn’t meant to under-value good lab skills, though.
• I have the obsessive sort of personality that once I get started on a project, I get completely and totally into it and don’t want to stop. I can be a bit of an obsessive work-a-holic from time to time. It’s difficult for me to disengage and I’m either completely into it or not into it at all. I suppose this is a good quality to have as a research scientist.
• I really like working with my hands in lab. Doing office work all the time is boring, and I think physically doing things with ones hands is sort of relaxing (sometimes; unless it's annoying).
• I have a good intuition for organic chemistry and bioorganic chemistry in terms of structure and reactivity. I just sort of know what reactions are reasonable, what structures are unstable, and have a solid sense of this in my head.
• I’m pretty good at spectroscopy, especially NMR. I’ve got a lot of chemical shifts, j-values, and solvent peaks for proton and carbon (well not j-values, but whatevs) in my head from doing a lot of synthetic organic chemistry, and correlations make sense to me. It’s the sort of visual puzzle that I’m good at, and I have a fair bit of experience with it. I’m also decent with MS, UV, and IR.
• I’ve got a decent knowledge base in a variety of fields. Along with knowing organic chemistry, I also know a fair amount of biochemistry and molecular biology, both the theory and the lab techniques. I’m kind of one of those hybrid interdisciplinary scientists, and this is just because I really do like it all.
• I'm pretty used to the idea that shit doesn't work most of the time in science. I guess I've spent enough time in labs to figure that out. Having shit not work doesn't really bother me that much--you just need to keep trying and hope that with hard work and a bit of luck it will all work out.
• It's pretty drilled into me to write everything in my lab notebook and label everything. One of my profs said that it was very easy to repeat reactions that I did and to keep up the good work. But my synthetic organic lab notebooks are a lot better than, say, the project I'm doing now where I'm never sure how much to include and there's a lot of "continued on page XXX" and MS spectra after MS spectra. My molecular biology lab notebooks are the worst, especially when I'm using a lot of kits.
  

AA structures

I recall last year a senior biochem major told me that she had forgotten the structure of the amino acids over the summer DESPITE THE FACT THAT SHE WAS DOING A THESIS ON PROTEIN BIOCHEMISTRY*. This horrified me. She was a great student, a hard worker, got good grades, etc. I guess people give memorizing structures a bad rep, but I think if you get a degree in biochemistry, you should fucking have the amino acid structures in your head, IMHO.

So today, I decided to test myself to see if I was being too judgmental. I needed to memorize the structures of the amino acids and the one letter codes last fall for structural biochem as well as the nitrogenous bases, so it's been several months since someone demanded that I have this knowledge at my fingertips. I guess took another biochemistry course after that, and I look at a lot of nat. product structures, so it's not like I just shut my brain off to biochem and peptides since I took that course. So I drew them all out from memory today just to test myself.

And I knew all of them still. All 20 of them right, suckers! Well, the one letter code for aspartate slipped my mind briefly, which is D, by the way. And I stupidly dropped a hydrogen drawing a couple of the bases. But I still knew which places had carbonyls and which had amine functionalities.

So phew. I guess I still feel justified in calling myself a biochemist, and in holding people to this standard. Is this an unreasonable standard? I dunno. I don't think so.

*okay, it was enzyme kinetics, so she wasn't exactly looking at her protein structures all the time--just dealing with bi-bi ping pong shit, but still. Still. Degree in biochemistry.