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Day at Work

Posted Jul 09 2010 6:32pm
This summer, I've been able to work for one of the labs at my university in an undergraduate research summer program. My lab is studying gene therapy, a subject that I am finding more and more fascinating as I learn more about it. The work is difficult, but not beyond my ability; and I'm working with a lot of other students, as it's a teaching laboratory and they're used to amateurs like me. I've been told that some graduate students go into research with no laboratory experience; but I'll escape that problem because it's my second summer now in the program and I'm finally getting the basics down. They're even paying me a stipend, which can work like a scholarship to help pay my tuition next quarter.

It's been three weeks now; and there wasn't any break in between finals and the start of work, so every day when I get up it's difficult to get out of bed. I'm not physically tired; but I'm mentally tired, and weekends never seem to recharge me completely. Thankfully, the lab is a relatively quiet, relatively orderly environment; and the people there don't seem to mind it when I take things literally or ask a lot of questions or lecture at them about something. So despite being tired, I'm doing well; and while my work isn't my absolute best, it's apparently acceptable, as I've only been reprimanded once (it was justified--I nearly damaged an experiment by absent-mindedly taking the cover off a cell culture; and afterward I was so embarrassed I almost cried.)

Today I worked on the last few steps of a process that lets us replicate DNA inside bacterial cells. It's really quite fascinating the way it can be done. The little bits of DNA we have--usually ordered from a company--are mixed into a bacterial culture (a strain of e. coli that's good for this sort of thing), and then the bacteria are heated for a little while to cause some minor damage to their cell membranes and let the DNA get in, and hopefully integrate itself into the bacteria's genome. Then, we let the bacteria multiply, over and over; and the DNA gets copied along with them. But only some of the bacteria managed to integrate the DNA we wanted--so, how to sort them out from the normal bacteria without our DNA in them? Can't very well go at the job with a very small set of tweezers and an electron microscope.

The answer is that the bits of DNA we wanted to replicate have genes for antibiotic resistance in them, as well as the genes for whatever we're interested in (at the moment, there's a lot of green fluorescent protein involved, because it's a great marker that you can see under fluorescent light if the cell's expressing it). So, when we dose the whole lot with antibiotic, any bacteria that have taken up the DNA we want them to copy survive, because when they had taken up our DNA, they had also gained antibiotic-resistance!

These survivors get to replicate again until there are a whole lot of them and the growth medium is cloudy; and then the bacteria are filtered out, and exposed to chemicals that first break their cell walls, and then put through filters that get rid of everything but the DNA. Eventually, the DNA gets cut into little bits by adding the restriction enzymes that will cut it where we want it (the original bits of DNA had, on each end, places where specific restriction enzymes "know" to snip the DNA).

That's the basic procedure. I'm probably leaving things out or not getting some right; but it should be roughly what's going on. The company that made the chemical-and-filter kit also printed a really precise protocol that tells you pretty much exactly what to do, given that you know basic lab procedures; and today I did a lot of it nearly on my own, only stopping when I had questions like, "Where do we keep the isopropyl?" or "How do you set this centrifuge?" I was proud of myself; because up until now I've not done too much by myself, except for easy things like setting up gels for Western blots (a way to sort proteins by size) or maintaining cell cultures.

And there's another thing I like about working at the lab. Scientists know how important it is to have precise instructions, because you've got to do things exactly the same way every time if you expect reliable results. There are protocols for everything, from mixing up buffer solutions (which is something a grade-school child could do) to doing really complicated assays that take weeks. And that means that I often have access to a written list of exactly the steps I need to take to do something--which is practically my optimum learning style! Granted, the lists assume you are a relatively experienced researcher; so I ask a lot of questions; and the more complicated things (which is most of them), I have to learn by watching someone else first to get all of the little tricks and things that the protocol expects a scientist would know. I carry a clipboard to make annotations on my own copy, so I won't forget.

There's a lot of small talk at the lab; but people seem to somehow know that unless you're working on something so easy that you don't need any brain for it at all, you don't want to be bothered when you're trying to concentrate. So I only have to practice small talk when I'm not working on something, which is okay. They aren't prone to the vapid sort of small talk that non-nerdy people are so fond of, and are generally quite interesting to talk to although, like most people, they are currently fascinated with the World Cup. (I can see the obvious skill and strategy involved in the game; but I am not too fond of watching competitive sports.) I think, if I have to practice small talk with anyone (and I do have to practice, as it seems impossible not to be understood by NTs unless one does make small talk), I'd rather practice with scientists and science graduate students than with anybody else.

Near the end of the day, I did viability readings on some of the cell cultures we'd been growing. This particular cell culture had been infected with a modified version of an adeno-associated virus, which is a beautiful little virus that can carry genes into the nucleus of cells. AAV is a helper-dependent virus, which means that it needs another virus to actively replicate (adenovirus works, and some others); and when it doesn't have that helper virus, it just sits around in the cell. But that's great for us, because the middle of the AAV genome (the part that lets it replicate and encapsulate itself into more little AAV viruses) can simply be snipped out and replaced with something interesting; so that when the AAV sees there isn't any helper virus and enters the nucleus, the gene we wanted gets expressed just like any other gene. In this case, it's green fluorescent protein we're looking for. The gene came from a jellyfish; and when the cell uses it, the protein that results glows a lovely green under fluorescent light--a great way to detect the cells where the AAV has made itself at home!

Today, on Day Seven after the cells had been exposed to our genetically engineered virus, the first bit of green glow started to appear; and I spent some time counting cells to make sure they were still alive and healthy. You do this by taking a little bit of the culture and mixing it with a blue dye that's poisonous to cells. Living cells will exclude it; dead ones won't care. So you count the bright cells against the blue background, under a microscope and in a known volume of dye-and-culture mixture; and you get a viability percentage. We had a lot of different test conditions; so it was a repetitive procedure--lots of little tubes of cells; lots of little bright cells on blue-dye background. I complained a little because it seemed expected not to like to count cells; but in reality, the only thing about it that I didn't like was that it wasn't something new to learn. I don't mind the repetition at all, especially because they let me listen to an mp3 player when I'm working on something (today, the audiobook was Peter Pan, which I love for its almost eerie combination of real danger and childish innocence). It's rather satisfying to do things exactly the same way every time; and my perfectionism doesn't hurt a thing when you're supposed to be trying to do things near-perfectly anyhow!

Despite a decent day at work, I was still tired out completely when I finished. I made a minor mistake in judging my remaining reserves: I had really wanted to visit the used bookstore; and I thought that I would be able to rest all weekend, so it wouldn't be such a bad thing if I got overloaded today. Well, I walked to the bookstore all right; but by the time I had gotten there, the heat had gotten to me, and I spent some short time on my knees in front of the clearance section, swaying and staring unseeingly at worn paperback romances and trying to conceptualize the idea of "get up" and "you're supposed to be looking for a book, you ninny!". I did find some books, though--a nice copy of Ender's Shadow; I, Robot; and Black Beauty, which I remember loving when I was a child. Dragging myself home was no pleasure. I don't actually remember most of it, except that I stopped halfway to rub my feet! But all that was soon repaired by getting home and being greeted by two affectionate, hungry cats and the prospect of a cool shower; and now I'm only tired, not overwhelmed anymore.
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