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:
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”).
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).
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.