My Master’s Project

All labwork is overseen by the disembodied head of Muppet lab assistant, Beaker.
All labwork is overseen by the disembodied head of Muppet lab assistant, Beaker.

I have begun my Master’s project in earnest and the goal is slightly different than what I’d been doing before.

First, I’ll repeat myself. I’m a biologist and I work with introns in C. elegans. C. elegans is a type of nematode worm that naturally lives in soil or on rotting vegetables. It is also one of the most widely used model organisms for biological research.

Worms! Ew! Gross!
Worms! Ew! Gross!

Introns are unused sections of genes. You’re probably aware that DNA is in our cells and contains the instructions for how an organism functions. The human genome contains around 25,000 genes and those genes are split into two parts, introns and exons.

Exons are the part of that gene that are actually used to produce things in your cells, while introns are spliced out and removed. So why are introns on there at all if they’re removed?

Well it turns out that some introns increase expression of the genes they’re in. My project looks at how placement of those enhancing introns affects expression.

Experiments in plants have shown that an enhancing intron works best when it is placed near the start of a gene. Experiments in C. elegans have suggested that, but no experiment has outright proved it. My project will hopefully do that.

I’m measuring the expression of genes according to how introns affect them, so I get to pick which gene to use. When picking a gene like this scientists often pick what are called reporter genes. The expression of these types of genes is easy to measure, often because they have produce light or fluorescence of some kind. The light tells you whether the gene is on, but also at what level it is turned on based on how bright the cell is.

Previously I was using a reporter gene called GUS. GUS is an enzyme that digests a specially prepared sugar, releasing a blue chemical that was attached to that sugar. The blue chemical is then visible to the naked eye.

There were a number of problems with that experiment though. First, adding the sugar chemical to the worms was a pain, taking about three days to set up and look at. Plus, the blue color was difficult to measure precisely because most of the machines in the lab are set up to measure red or green colors, not blue. Finally, GUS is traditionally a reporter gene for plants, not C. elegans. This could’ve been introducing other problems that we couldn’t easily identify. Thus the use of the GUS reporter gene has been scrapped in favor of another reporter gene.

I’ll be using Green Fluorescent Protein (GFP) as my reporter gene now. GFP is widely used in C. elegans and many other organisms. The protein created by the GFP gene glows green when you shine a red light on it. Very easy to see and measure. None of that three day procedure for GUS. I just pop the worms under the light and take a look.

Why weren’t we using this procedure before if it’s so easy? Two reasons!

Reason number one: C. elegans won’t express GFP without introns in the gene. So does that mean we proceed and hope one intron is enough or do we add the standard amount of introns to get expression? I’ve decided to see what the GFP looks like with the standard introns scientists put in it for C. elegans and without them. I’ll also be testing with an added intron. The whole thing is a little complicated so here’s a diagram to explain.

Here are the constructs I've been creating. The wide parts are exons and the thing parts are introns. The green bands are the GFP which will glow green in the worms. The white bands are a scaffold which allow the worms to express GFP
Here are the constructs I’ve been creating. The wide parts are exons and the thing parts are introns. The green bands are the GFP which will glow green in the worms. The white bands are a scaffold which allow the worms to express GFP.

There are eight different constructs I’m making. They are a combination of three different features that are present or not. Are there introns in the first GFP? Yes or no? The second GFP? And is the Unc54 intron there? This allows us to control for the positional effect the standard introns in C. elegans GFP.

Reason number two: Those eight constructs above? Those aren’t made yet! All the GUS constructs were made when I started the project. I’ve been working on making the new constructs for a few months. It could take a few more months to finish.

So my project is to make those constructs, put them into worms, and then see what the worms look like. As I perform these steps I’ll make more posts about what work I’m doing in lab and why its so cool.

-Mister Ed

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The Fault in Our Stars

I read The Fault in Our Stars next to my cat, Carmelita.
I read The Fault in Our Stars next to my cat, Carmelita.

This weekend I read a popular young adult fiction book, The Fault in Our Stars by John Green.

The book is written from the point of view of Hazel, a teenager with lung cancer (15% survival rate, likely higher for her specific case) who is always hooked up to a respirator.

She begins attending a support group for teenagers with cancer. At the support group she meets Isaac and his friend, Augustus.

Hazel begins dating Augustus, who has a a less lethal type of cancer than her (osteosarcoma, 80% survival rate).

The book explores how teenagers react to their own terminal illnesses, how their families and friends react, and what a cancer patient might want to be remembered for.

One of Hazel’s struggles is that she doesn’t want to only be known for having cancer.

She loves poetry and reading. The book acknowledges that she is even smart enough to be attending community college for some type of English degree.

Unfortunately, all of Hazel’s friends from before cancer only see her as a sick person, not someone who loves literature.

Sick of pity from her old friends, Hazel has withdrawn from public life and only interacts with her family and her new friends from the support group who personally understand her illness.

Hazel also withdraws because “she is a grenade.”

She fears her eventual death and doesn’t want to hurt anyone who gets attached to her. Thus, she avoids making such attachments.

I’d definitely recommend the book. It’s a quick read, well written if predictable, and on a topic worth learning more about.

There’s also a movie adaption coming out on June 6th 2014 in the USA. The book wasn’t enough, so I’ll be going to the movie to get more even if the story is the same.

I’ll probably check out more of John Green’s work too.

-Mister Ed

Injecting Worms

This is what my computer captures under my microscope when I inject a worm.
This is what my computer captures under my microscope when I inject a worm.

I gave an extra post about one of my jobs. It seems fair to cover the other job as well at some point!

I study introns in C. elegans worms, but how do I get the specific introns in the worms?

I need introns in specific placements in specific genes in order to study them with scientific accuracy.

The gene we are studying is simple. If the worms are put in a solution called X-gluc, they turn blue.

Based on where our enhancing intron is in the worm we expect it to turn more blue if the intron is closer to the start of the gene or less blue if it is near the end.

So I have these genes that I’m putting into the worm. They get in by injecting them like you see in the picture.

The needle of DNA is aimed at the gonad of the worm.

C. elegans worms are hermaphrodites. They contain sperm and eggs and they self-fertilize.

The worms are “male” at first, producing a bunch of sperm.

Later on they produce eggs and then they fertilize their own eggs with the sperm stored in their body.

Since they contain both genitalia the whole area is referred to as the gonad.

I aim my injection at the gonad, hoping that the DNA I’m injecting will get into the fertilized eggs.

Then the injected worm is put on a plate with lots of food and I hope that its babies will have the injected DNA.

But I don’t test for “blueness” immediately.

When a worm is first injected, the DNA is inside its cells, but not necessarily integrated into the cell’s chromosomes. I need the DNA to be a part of the chromosomes.

There are only two genes in the mix of injected DNA that will integrate. One gene is the blue gene, called GUS. The other gene is called unc119.

Unc119 is to “recover” the worms.

The worms I inject lack unc119, which is a normal gene for worms.

In a natural wild-type worm unc119 aids the development of the worm’s neural network. Without it, the worm has poor neural connections and has a lot of trouble even moving around and eating.

So the first way I test a successful injection is by looking to see if the babies of the injected worm are moving around normally or flopping around.

The normally moving ones were successful and now have unc119. They are “recovered” back to their natural wild-type state.

The floppy crippled ones did not have a successful injection. Either I missed the gonad, I didn’t inject enough DNA, or the eggs that got my injection didn’t fully germinate.

There are other markers I use to see if an injection was successful, but I’ll get to those later!

-Mister Ed

Good and Bad

I had a strange amount of ups and downs today.

I started off by trying to install some child locks on our bathroom cabinets. Our new cat has been trying to sneak in and we think it’d be best if she doesn’t have access to all the toilet paper at once or all the chemicals that could hurt her under the sink.

Child locks come in two parts, the part you have to push to open it and the part the pushy part inserts into to keep the door locks. I’d glued the holdy part onto the cabinets on Monday.

I checked the glue today and was a bit rough in handling one of the holdy parts. It popped off in my hand. I sighed and reglued it on.

I glued on the pushy part and found it was very difficult to align it correctly with the holdy part. Fearing that I did it wrong, I only glued in one of the pushy parts today to see how it looks tomorrow.

After that frustration I went out to fix the flat in my bike. The flat had happened on Friday and I got new tubes on Monday.

I put the first tube in and it immediately popped. Figuring they can occasionally be duds, I pulled it out and put a second one in. It seemed fine and I biked off to work. It popped just before I got onto campus. I sighed and walked the rest of the way to my job.

I figured I was too pissed off to immediately get to work at lab, so I decided to make a quick appointment with an optometrist in town.

The optometrist informed me that I needed to know the exact amount of coverage my health insurance gave me for vision before I could get an appointment. On to calling the health insurance company then!

I called my health insurance and was put on hold (typical). I waited and zoned out not listening to the recorded message. Then the message said, “Goodbye,” and it hung up. I have no clue what led up to it saying goodbye, but I’m pretty sure it wasn’t the usual hold music.

I called again and avoided the previous menu options that had resulted in the robot disconnecting me. I ended up waiting an hour on hold before I got to talk to a human person.

The whole time it faked me out by switching up which robot voices were telling me about the health insurance website. Each time the voice changed I thought I had finally reached a real person only to have my hopes destroyed.

I eventually got the health insurance and the optometrist appointment set up.

After that my day improved. I stained some worms to see if they turn blue over the next two days. I checked some injected worms to see if the DNA I injected into them had successfully integrated into their genomes. IT HAD! WOOHOO!

I called my wife to come pick up my bike during the afternoon so I wouldn’t have to walk it home. She came by and put it in the car and reminded me why I love her.

At the end of the day my wife came home with two cupcakes for me.

A good day in the end despite the rocky start! 🙂

-Mister Ed