One of my favorite protocols in the lab is running SDS-Page gels. Boss thinks Im nuts (I guess other people hate running them), but I kick ass at them so he lets me be giddy.
The all knowing Wiki proclaims that SDS-Page is: a technique used in biochemistry, genetics and molecular biology to separate proteins according to their size (length of polypeptide chain or molecular weight).
We're trying to separate out three proteins HIV makes-- gp160, gp120, and gp41. Gp160 is just a precursor protein, it gets chopped into the two bits that HIV really wants: a transmembrane protein gp41, and gp120, which noncovalently bonds to gp41 to create the spikes that stick out of HIV that attach to your cells.
(from 'Molecules of HIV')
The thing is, when we blow up the cells in our Pulse Chase experiment, we collect all the proteins the cells make, not just the HIV proteins. So we do something cool called an immunoprecipitation-- we add HIV antibodies to the cell goo, the Ab stick to the HIV proteins, and we add beads to the goo that stick to the Ab. When we wash everything off, only the beads, the Ab, and the HIV proteins are left! Then we boil the samples to denature the proteins (they need to be linear to run in a gel) and BAM! Pretty gel time!
Sunday, October 29, 2006
One of my favorite protocols in the lab is running SDS-Page gels. Boss thinks Im nuts (I guess other people hate running them), but I kick ass at them so he lets me be giddy.
Thursday, October 26, 2006
Wednesday, October 25, 2006
Monday, October 23, 2006
This weeks paper covers Streptococcus—the bacteria that gives little kids alllll over the world strep throat every winter. It likes to invade your tonsil cells, or sometimes cuts and wounds. They like epithelial cells. Well bacterial infections in your flesh are never a good thing, but in the mid 1980’s, something strange happened. No, not just the rise of fundamentalist Christianity in the US, strep infections were killing people, through a different kind of toxic shock syndrome, and through necrotic fasciitis. Do NOT Google necrotic fasciitis after you eat. Or before you want to eat. Seriously.
So what happened? A sweet, sweet lesson in evolution. Strep stole a gene from a phage (yes, a bacteria stole a gene from a virus), and it learned a new trick:
Emergence of a bacterial clone with enhanced virulence by acquisition of a phage encoding a secreted phospholipase A2
M3 strep infections have a higher death rate than other strains of strep. So these guys compared genomes of strep collected over the past 60 years, trying to find out what was so different, so deadly about M3. They found that M3 had stolen a phospholipase A2 gene from a phage virus (arch enemy of the bacteria) called SlaA. SlaA is the same stuff that you find in Australian brown snake venom. Yeah. They aren’t connected, but its still cool. Hehehe!
So these guys wanted to be sure they found the ‘right’ different gene in M3. So they went through a series of simple experiments to answer some basic questions:
1—What does SlaA do if you put it on tonsil cells?
Okay, easy enough to answer! Put some purified SlaA proteins on a culture dish of cells, watch what happens!
….. Nothing happened.
2—Okay… So what happens to the bacteria if you take away its SlaA gene?
They took away the M3s SlaA gene by making a delta-SlaA mutant and threw it on some cells! The bacteria behaved ‘normally’—they infected the cells, but not crazy nuts killing everything like the wild-type M3. So they were on to something! More experiments!
2A—Take a wt M3, but add SlaA antibodies to block it from doing anything. The ‘wt’ M3 acted more like a delta-SlaA mutant (missing SlaA)!
2B—Take a delta-SlaA mutant, and add exogenous SlaA to see if you can restore its phenotype. Sure enough, mutants complemented with SlaA proteins started killing more cells!
3—Well what the hell is going on??
When M3 strep hits saliva or tonsil cells, it starts secreting SlaA and gets it into the cells (remember, exogenous SlaA on its own didn’t do anything, it must act within the cells). SlaA works inside the cell to increase M3s ability to adhere to cells, and it itself toxic. They still need to work out the details (how, exactly, does SlaA get in? what does it do when it gets there?), but they’ve got a good case that in one fell swoop, this strain of bacteria stole a gene from a virus and became more virulent. This isn’t the *first* time this has happened (look at your own genome!) and evidently has had a huge impact on the evolution of life on this planet.
Tuesday, October 17, 2006
Well, that headache went away. I guess that means I dont have a colony of Staph eating my brain. **sigh** Oh well. hehehehehe!
As promised, I read through one of Dr. Kielians papers:
The role of Toll-like receptors in CNS response to microbial challenge
Sure they do a better job of explaining their research than I can, but heres my summary anyway--
There are these great cells in your brain-- astrocytes and microglia, that act as 'gatekeepers.' They keep bad stuff out. But sometimes, despite their best efforts, something does get through. So theyve got these receptors in their membrane that detect 'bad' stuff floating around called 'Toll like receptors.' Lots of families and lots of kinds of these receptors that non-specifically detect foreign stuff floating around your brain: Double stranded RNA (a virus got in!), PGN (a bacteria got in!), that sort of thing.
They then proceed to freak out.
When they detect something foreign has gotten past the blood-brain barrier, they start the inflammatory response, releasing cellular messengers that call white blood cells to the brain (thats why the ball of bacteria in your brain will also be filled with puss. YEAH!). One of the problems with a Staph infection in the brain (other than the fact Staph is in your brain), is that long after the bacteria are dead, their body parts are still floating around. The astrocytes and glial cells think that means there is still bacteria eating your brain, and they call up MORE white blood cells. This causes 'bystander injury,' where normal neurons are killed because of an over-active inflammatory response.
Dr. Kielian is hoping that their characterization of this pathway with Staph can help us better understand other diseases like HIV dementia and MS, which might be caused or propogated by this Toll receptor response.
Im going to think of this every time I get a headache now. Ugh.
Monday, October 16, 2006
Im going to be a PhD student in microbiology next year. Weird problem—Ive never taken a course in microbiology. Really! It was a low level course in college, and I didn’t want to take it for an elective when I could take a higher level (aka more interesting) course instead. So I just went straight from general bio courses to virology. I kinda suck when it comes to bacteria, or fungi, or amoebas, or any of the other cool critters out there.
So once a week I want to talk about something that’s not viruses. Very, very hard for me :) But I got to hear this speaker today that was SOOO COOL: Tammy Kielian!
First of all, she was just a kick ass chick—she had a family, a ton of $$$ in grants, and she was a great speaker too. But what pushed her into a whole new level of cool was her research on Staphylococcus aureus. You might have heard it referred to as ‘Staph’ before from a news program—‘Staph’ is infamous for being a problem in hospitals, causing ~12,000 deaths a year. If I recall correctly, that was the official cause of death of one of my grandmothers. Dr. Kielian studies a really weird thing Staph will do… Get into your brain, kill some tissue, and form a big, puss filled abscess.
Yeah. It doenst just get in your brain an obvious way, like a dagger to the forehead—it can contaminate the brain from a dental infection or a sinus infection, or it can spread if a chunk of bacteria dislodge from a main infection site (in the lungs, wherever) and gets stuck in the tiny blood vessels in your brain. Im going to try to read one of her papers tomorrow to give you all the gory details, but one of the major problems of this kind of infection is that its hard to diagnose. Symptoms don’t show until enough brain tissue dies to screw up your motor skills/memory/speech/etc. And the thing is, its not 'that' uncommon. Its not really common like the flu, but evidently a big ball of bacteria in your brain isnt all that rare either! AAAHHH!! **shudder** But one of the few ‘first signs’ is headaches… and Ive got a killer headache today. And Ive had a runny nose since I moved down here. Sure Ive never had a sinus infection, but still, I just know I have a hole in my brain filled with staph and white blood cells and blown up neurons. I just know it.
More to come!
Wednesday, October 11, 2006
Ugh this post is really not going to be honorable enough or eloquent enough to warrant the Sagan reference in the title, but I think its appropriate.
When I was little, I wanted to be an astronaut. I loved Star Trek, I loved Star Wars, I wanted to explore new worlds, and I thought space was the only place I could go for that. I went to Space Camp a couple times, took a course on astronomy, just in love with all of it. The idea of doing things and seeing things that no one had seen before.
I decided my path to space was going to be through the Air Force Academy. If most astronauts were in the military, then I was going to join the military. I worked my ass off to get into their ‘Summer Scientific Seminar’—if you got into that camp, you were for all intents and purposes guaranteed a spot in the Academy for college. I got in. Only two people from every state got to go, and I got in. The path was laid for ERV to be an astronaut!!! WHOOO!!!
So what happened? I dunno. Through a series of fortunate events, I grew disillusioned with the USAFA. I didn’t fit in with the other students. I didn’t fit in with the atmosphere of the university. It wasn’t ‘right.’ I grew disillusioned with astronomy. Nebulas are amazing, but I was never going to get to fly near one. I was never going to put my feet on another planet. All space could offer me was cold. By the time this all hit me I was graduating high school and I needed to make some decisions about my future, so I decided to go into biology. I was meh about it at first. Honestly, I just did it for med school. What else could you do with a degree in biology?
BWAHAHAHAHA! Again, though a series of fortunate and completely unintentional events—I found a way to explore new worlds! I realized a couple days ago that the reason why I love my job so much is that Im getting to live out my childhood dreams, though I thought they were killed years ago by impossibility. No one else on the planet is doing what Im doing (well, except my boss)—Im exploring a part of this universe that none of us have seen before! People are depending on my observations to make a map to explore other parts of the universe! And to be completely trite—this universe is right in front of our faces, not far far away.
And Im not the only one doing this! Scientists all over the planet are busy exploring, discovering, observing things no one has seen or understood before! As Sagan put it, we’re candles lighting the dark of the universe. And right now I friggen feel like Im dancing around a bon fire (sure we’re still surrounded by darkness, but all of our candles put together looks real bright right now). I know I suck at explaining what I do to my friends. My parents don’t even ask what I do any more—its just “Are you enjoying work? Gooood!”. But I really really do try to light other peoples candles. I try to get them excited about science, whatever theyre interested in.
Yet some people don’t want their wicks lit. They want to live in the dark, or at least they think they do. I cant say why, I cant even imagine. But a group of the leaders of the Dark are most definitely the Creationists. They work so hard to keep their followers in the dark, some spending their lives dedicated to extinguishing candles. They teach those with extinguished flames to fear the light of science. Or delude themselves into thinking their ignorance is a camp fire. But I think this methodology is finally catching up to the leaders of the Creationist movement.
Recently, as I posted about, the discovery of RNAi won the Nobel Prize in Medicine… Go to Answers in Genesis, the YEC go-to site. Search for RNAi. Go to Harun Yahya, the Islamic counterpart. Search for RNAi. Go to ICR. Go to ARN. Go to DI.
WHY ARENT ANY OF THESE PEOPLE TALKING ABOUT RNAI??????
Im about to start using siRNA myself—its absolutely necessary for my PhD project. Im dancing RIGHT up against the flames of science right now, and using siRNA and evolution. The leaders of Creationism are so scared of science that they cant address modern science! Second law of thermodynamics, they feel safe with. Evolutionists are evil, they feel safe with. Eyes are too complex, they feel safe with. But mention some new world that they haven’t even imagined, some fantastic mechanism they didn’t even know existed, and theyre scared to death to even address it.
Just a very strange observation.
Tuesday, October 10, 2006
Wednesday, October 04, 2006
Its weird—what Im doing right now in the lab is exactly what I was doing this time last year. But this time last year I was a retard that didn’t know how to pour an 0.8% agarose gel. Seriously, my boss had to show me how to do that (and he was very sweet about it, btw. Didn’t even laugh when I tried to ‘write down the protocol.’ **blush**). Now Im doing these insane experiments by myself.
Including the dreaded 12hr time-point. Ugh. No matter how early you start this protocol, the 12hr point is always in the middle of the night!! ARRRG! I go to sleep 30 minutes ago!
So Im typing this blog entry to keep me awake until I have to drive to work and blow stuff up for 45 minutes. Yes, blow stuff up. Blow radioactive stuff up. I said these experiments are insane!
I infected some cells with a virus a couple days ago. Let the viruses get real comfy—let them set up shop, getting the cell to make its proteins for it. Hehehe But this morning, I took away all the cells’ food. I starved those bastards so they couldn’t make any proteins. Hehehe Then I gave them food, laced with radioactive Methionine and Cysteine amino acids!! Methionine and Cysteine are the only amino acids that contain sulfur, so we just exchanged a regular sulfur atom with a radioactive S35 atom—that way we could find out exactly what proteins the cells were making, because all the proteins they make will be radioactive! And those cells are making viral proteins :) We wanna know how much of a certain viral protein is getting made, and how fast its getting chopped into two pieces that the virus really needs to stay infectious.
Well we can collect all the proteins the cells make by blowing them up and collecting their innards (my favorite part, personally hehe). We blow them up right after we give them radioactive food, an identical batch of cells 2 hours later, another one 4 hours later, etc etc etc up to 24 hours. Again, we want to know how fast this protein were interested in gets chopped into two, so we take a snap-shot every couple of hours.
We can pull out the viral proteins we are interested in by making them stick to beads with special antibodies. These proteins get run out in a gel to separate them out by size—put a piece of film on the gel, let it set for a while, and the S35 excites the film when exposed to light, and you get a sweet ass gel that you can get lots of AWESOME data from!
… But that will be like, a few weeks from now. Sorry. :(
Monday, October 02, 2006
Sweet! I remember when I first learned about siRNA—Fall of my senior year of college—Molecular Genetics—‘Control of Leaf Morphogenesis by microRNAs’.
As hard as my professor tried, I just didnt 'get' it at first. RISCs, Dicers, Pri-miRNA, Pre-miRNA-- The whole process is elegantly insane! Single stranded siRNA goes out into the cell and binds to its exact complement creating a double-stranded RNA. Double-stranded RNA is a magnent for this complex that rips the dsRNA apart. Its one of the ways your genome keeps all those endogenous retroviruses quiet ;)
This animation explains it much better than I can!
Isnt it nuts!! No wonder it took us so long to find it!
Its turned out to be not just an interesting process, but an absurdly useful tool as well. Plants, animals, insects, fungi, viruses, everybody has non-protein-coding sequences in their DNA that code for silencing RNA-- so that means that we can design an artificial 'siRNA' for a gene we're interested in, in basically any organism, and see what happens when we make the gene shut up!
Its so much more practical than mutating a gene so its not expressed all to see what happens. Sometimes when you totally knock out a gene, the organism cant even develop right, so youre no closer to understanding the gene fuction than you were before. But again, with siRNA, you can let the organism grow up and only silence a gene to see what happens, and then when all the siRNA is degraded, you can see what happens when the gene fuction is restored! Its a natural process that we can harness.
But Im kinda sad my pick didn’t win. I was partial to Roger Tsien because one of my research projects is all about making things glow. Tsiens lab made a billion new fluorescent proteins:
Well, I appreciate the ability to make my viruses in Skittles colors.
Sunday, October 01, 2006
IVE GOT A NEW PUPPY NEPHEW!!!
Bro picked Baby Duke up at a Humane Society adoption drive! 10 month old yellow lab, sweet disposition, and at a shelter. **sigh** My family cant go to adoption drives/shelters/etc without bringing 12 dogs home, but for some reason my bro thought he outgrew that. He didnt. hehehehehehe!
The thing is, bro already has a dog-- Miss Speckles. Miss Speckles was a street dog before she found bro. She doesnt *like* other people. She doesnt *like* other dogs. Luckily bros house is like 8,000 square feet, so the two pups have avoided confrontation so far.
HEHEHEHEHEHE I GET TO PLAY WITH A PUPPY OVER CHRISTMAS BREAK!!!