Smooth Sailing!

Finally I’ve had a productive week where things worked!

I made RNA, made DNA, and verified that I can stain extracellularly with my antibody!

Which sounds like gibberish (or near gibberish), so here’s the breakdown:

Making RNA
I’ve mentioned before that one big advantages to using Xenopus laevis (frog) embryos for developmental research is that the eggs are huge, so we can inject them with things.

Here’s an image (just pulled from google) to show that. On the left you can see a glass tube, which is actually a needle. We use a machine that heats up the glass tube and pulls it to be a certain diameter. You can then stick that (using a device called a micromanipulator that holds the needle for you and can be moved along multiple axes by turning dials).

And my current main experiment involves injecting a morpholino (which is a man-made chemical similar to DNA or RNA). But you can also just inject DNA or RNA.

Quick reminder that DNA is transcribed to make RNA, and then the RNA is translated into protein. And the proteins are what actually do all* the stuff in your cells.

*Well, they don’t do everything. RNA actually can do some things, but proteins are the bulk of the actors. They modify molecules and other proteins, move molecules and other proteins, and bring together molecules and proteins in specific patterns.

So if you inject DNA or RNA, the cell will actually work with it and make stuff from it. So if you inject DNA, it will make RNA from it, and then make protein from that RNA. And if you inject RNA, it will make protein straight from that.

There are some advantages to injecting RNA instead of DNA (basically you get more cells actually making the protein), so I wanted to make some RNA that will encode a red fluorescent protein called mCherry.

Okay, I started explaining how exactly I did that, but quickly realized that to really explain it would mean explaining a lot of background concepts, and it’s Friday afternoon, so I want to go hang out with people instead of sitting here writing about molecular biology for several hours, haha. But basically you can put your DNA, RNA nucleotides (basically RNA legos), and the proteins that can put together the RNA legos to build an RNA strand based on the DNA all together in a test tube, let it sit for several hours at 37°C and magic happens! And then you purify out the RNA.

The real trick with making RNA is that your body actively destroys RNA. You secrete a protein called RNase. RNase chops up any RNA it encounters. And your skin cells, hair, sweat, tears, mucus, saliva etc all have a ton of this stuff. Which means if I breath too hard on my RNA, it could all get degraded! So I have to be very careful (no talking, clean the whole bench down with a spray called RNase-Away, use gloves and change them after touching anything else).

But I succeeded! And now I have some mCherry RNA to play with (and by “play”, I mean “use for important experiments”).

Making DNA
Again I’m not gonna actually explain too much (sorry!), but basically I didn’t have enough of another DNA and wanted more of it. In biology we use E. coli to help us out in these situations! E. coli will take up DNA from their environment under the right conditions (which we can easily create in the lab). So I just had to get them to take up my DNA, grow them up in some bacteria-friendly, nutrient rich liquid, and then purify out my DNA.

So I’ll explain a bit about how I get the DNA I want and not a bunch of E. coli DNA, because that’s pretty cool. Basically, the DNA I want is in a little circle of DNA called a plasmid. Bacteria typically have lots of plasmids (which is actually how they can easily acquire resistance to antibiotics – they can trade plasmids with other bacteria, so if they meet and mate with a cell that has developed antibiotic-resistance, it can acquire the trait by taking up those antibiotic-resistance genes). But the ones I’m using do not. And the rest of the E. coli genome is on a much larger strand of DNA (called genomic DNA because, well, it’s the stuff that’s actually in the genome rather than these extra plasmids. In the picture below it’s labeled “Bacterial DNA”).

After I’ve grown up a bunch of bacteria that have my plasmids in it, I lyse the cells. Lysing is basically breaking them apart, so I disrupt their outer membrane so all the DNA is now just in solution in my tube (along with everything else). To separate my plasmid DNA from the genomic DNA, I take advantage of the fact that my plasmids are little and the genomic DNA is much larger. I can put my tubes in a centrifuge, which basically spins them incredibly fast (13,000 rotations per minute), which separates everything based on size (the biggest things go to the bottom). So now all the big genomic DNA is at the bottom, while my plasmid is at the top. So I can pull off the solution that has my plasmid in it and purify it from there. (The purification at that point involves a filter that the DNA binds to).

Antibody Stuff
I’ve talked about antibody staining before. But basically I can use an antibody that binds to my protein of interest to label it. I add on the antibody and it binds to all my protein, and then I add a fluorescent antibody that binds to that first antibody. And normally when we do this in fixed tissue (fixed meaning preserved, like formaldehyde) we put the cells in a solution that causes the outer membrane to become porous, so the big antibodies can get through. But I wanted to see what of my protein was imbedded only on the outer membrane, and not what is actually inside the cell. Fortunately, the part of my protein that my antibody binds to is a bit that sticks out on the surface of the cell. So in theory I could skip the step where we permeabilize that outer membrane, so none of the antibody would get inside the cell, and I could see only what bound to my protein on the outside.In theory it would work, but I wanted to actually try it out before incorporating it into my experiment (just because I don’t want to do a ton of work just to find out my detection method is faulty). And it does work! Unfortunately I had some technical issues (i.e. I destroyed almost all the cells from two of my three slides), so I’ll certainly be repeating the experiment. But at least I know it’s something I can do, so I can rely on that in the future if I want to!

So that’s been my week! And I’m all done with classes for the year, so I’m suddenly so much more productive in lab. Which is awesome because that’s what I actually want to be doing. While classes are certainly valuable, I’m just ready to be done. And I’ve finished all the classes required for my program (yay for being in a program with low class requirements!). That doesn’t necessarily mean I won’t have any more to take (my thesis committee will decide that when I meet with them in August), but at least it will probably only be one or two more, so I’ll almost certainly no longer have class every day of the week.

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Scientific Writing

I’ve started reading the blog Serial Mentor by a PI at University of Texas Austin, and he’s got some awesome (though much more science- and academia-specific posts than most of you probably care about). But he has a great post about the problems with academic-style writing and how to improve your writing.

And having just written a final paper and final exam for classes, it was really obvious how much I’ve ingrained a lot of the “correct” scientific writing practices that are actually terrible. Actually when I was writing my exam, I realized I had a sentence that really should have been about 4 sentences. I ended up crossing it out and restarting because it was so unweildy I couldn’t end it. And I am the master of sentences so long that they seem like they’re run-ons even though technically they’re not, haha.

But I’ve also been thinking about how scientific language is an impediment to public understanding. I remember in undergrad, reading a scientific paper was really difficult. And part of it is that there are a lot of terms you don’t know, and the more you get used to hearing certain words, the easier it gets. But the sentence structure that’s common is also incredibly difficult to understand. Usually there are prepositional phrases on prepositional phrases, so it’s hard to figure out what’s actually happening (as serial mentor’s post points out).

Which is funny because the goal of scientific writing is supposed to be clarity. That’s the reason it should be different from literary writing: you want to get to the point clearly and concisely. But that’s not at all what actually happens when you use passive voice and prepositional phrases for days.

Another thing I’ve been thinking about related to science writing and communication: in science we tend to be really careful about our word choice. There are very few things we can say with any certainty, so we carefully hedge our sentences to be clear that “the evidence suggests” or “these findings would indicate” and avoid saying “this is the way it is” because you can never prove anything with 100% certainty. Additionally, we often use highly specific terms because even small variations in the way an experiment is done can make a big difference.

But while these differences are very important in a research context, they’re pretty useless for explaining to the general public. The general public doesn’t care about details, and including them can make things more confusing than it’s worth. I think that sometimes explaining things a little bit wrong is better than being technically correct but having nobody understand.

So I’ve been trying to incorporate a great improv technique into my explaining: “yes and.” For those who haven’t done improv before, the idea of “yes and” is that you never shoot down somebody’s idea, because it shuts everything down and makes for terrible comedy. For instance:

“I’m an elephant! *flails arm-trunk*”
“No, you’re a cat.”
“Oh, umm… I guess I’m a cat then.”

isn’t a good way to start a scene. But this is:

“I’m an elephant! *flails arm-trunk*”
“Yes, and we’re on our way to Pride Rock to meet Simba!”

And I think this technique is also super valuable for explaining complicated concepts, and I’m trying to be better at incorporating it in my explanations (as well as the rest of my life). For instance when I took physics, I had what I thought was a very logical realization: because electrical currents generate magnetic fields, what made magnetic objects magnetic was that the electrons moved around atoms in a synchronous way that generated a magnetic field. And when I asked the TA about it, his response was basically “Well, not really, but you haven’t taken enough math for me to be able to explain it to you.” Which just left me more confused. And what I think would have been more valuable would have been him saying, “Yeah, that’s a good way to think about it! And here’s this extra piece of knowledge to build off of that.” In that second version, I would have learned something new from the interaction instead of just feeling like physics was a confusing and impenetrable subject.

So I want to be better at saying “yes and” as well as writing and communicating in ways that are clear, instead of just trying to be technically correct or sound official.

Why do I love grad school?

Two weeks ago I talked about how I’ve actively chosen to not to live and breathe only grad school (and actually I forgot to do a post last week because I was at a dance convention). But obviously I’m here. And this is absolutely where I want to be. So today I’m gonna talk about why.

First: I’m a question-asker. I have been given multiple nicknames based on how many questions I ask. From several completely distinct groups of people. I ask a lot of hows and whys. Which pretty clearly lends itself to scientific research. I just really like figuring out how things work.

Second: I’m a learner. I’ve always been that kid who enjoyed classes (yes, I even enjoyed high school). I love learning new things, and much of my free time is spent doing just that: I have historically jumped from hobby to hobby fairly often, and I think a lot of that is that I like picking up new things because there’s so much to learn when you start something new (For instance I’ve taken lessons in 7 instruments and mastered exactly none of them). So to basically get to spend 5 years being paid to learn about things I find really interesting and important? Pretty good deal.

Third: I’m a problem solver. I love puzzles. I love logic puzzles, I love crosswords, I even love 5000 piece puzzles of lighthouses. And solving research problems is like all of those things times a million. It’s like having a 5000 piece puzzle where you have to critically analyze each piece to make sure it even belongs in the puzzle and that the manufacturer didn’t accidentally make it the wrong shape. And other people have put together the top left corner, so you have to find that so you don’t redo that whole section that’s already known. And you may have a vague idea of what you think the picture will be of, but you may be entirely incorrect. Which would be an incredibly frustrating puzzle and would probably sell terribly. But while research is absolutely frustrating (guess who gets to redo two thirds of what they did this week because it didn’t work??? me!!!), I can get bored easily, so having a difficult problem with twists and turns is pretty much perfect for me.

Fourth: I’m a crafter. I actually love the physical actions of doing cell biology research. I like dissecting and I like pipetting. I knit and sew, both of which involve lots of careful planning followed by lots of repetitive, somewhat tedious actions. Which I actually really enjoy. The simple physical actions can be a nice break from the creative problem solving. And the more difficult ones (I keep getting too many muscle cells in my spinal cord dissections) are something I can practice at to get better.

Basically grad school (and cell biology research more generally) incorporates a lot of aspects that really appeal to me. I have yet to hear about a career path that does that as well (while I occasionally daydream about being a woodworker or pattern drafter or dance teacher, they all have 1-3 things I’d love and 20+ things I’d hate). So even though I don’t want it to be 100% of my life, I really love that it’s a large chunk of it.

Also sorry this sounds like a grad school application/interview, haha. It’s hard to talk about why I like grad school without it sounding really cheesy. Also I will say that even though I realized during undergrad that I would enjoy grad school, I didn’t decide to actually apply until I thought more about what career I wanted and whether I needed grad school to get me there. And I eventually realized that I wanted a career where I could be doing the project planning and critical thinking and literature analysis, and that meant I needed a PhD.