Cool Stuff from Last Semester

I did some cool stuff last semester in my science classes that I’d like to show you guys.

The gist of it is… This picture:


This is a picture taken by my lab group in my basic lab technique class last semester of a mouse fibroblast cell moving into a simulated wound on a glass slide.

Fibroblast cells are kind of like the contractors of your body when you get a scratch or wound. There are your first responders to the “disaster,” your immune system, and then fibroblasts go in to start the process of rebuilding your tissue by laying the foundation for other cells to move in.

A lot of scientists are interested in wound healing. How can we make it faster? How can we make it better so people don’t have lingering problems after the superficial injury has healed? How can we prevent infection? How can we prevent scarring?

Those questions are tested with a variety of experiments but one of the msot common is the scratch assay.

A bunch of fibroblasts are grown on a glass slide until they practically cover it. Then the slide is scratched.

The fibroblasts move into the scratch, thinking it is a wound. Their movement into the scratch is measured in a couple different ways and those measurements can tell us a little bit more about how wounds heal.

Which brings me back to the picture my lab group took. Obviously its got a lot of color and is very prety, but what are all those colors? What’s going on in that picture?

My lab group scratched the space above the big cell in teh picture. The cell is now moving into the scratch.

The red lines are called actin. Actin is the support structure of your cells. Cells move by extending actin filaments where they want to go and breaking them down behind them.

The green parts are called vinculin. Vinculin is spread throughout the cell and localizes into spots where the cell is attached to a surface to assist in adhereing to that surface. All those bright green spots are where the vinculin is helping the cell hold onto the glass slide.

The blue parts are cell nuclei. Each cell has one nucleus and I’ll bet you can pick out the one that belongs to all the actin and vinculin in the middle of this picture.

I did a lot more stuff on scratch assays in this class and leaarned a few new techniques, but the best part was definitely getting this picture.

Oh and apologies to any color blind people. I have no idea how to spearatae out the red and green things for you. Enjoy!

-GoCorral

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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

Liquid Nitrogen in the Lab

A thermos with some bubbling liquid nitrogen at the bottom.
A thermos with some bubbling liquid nitrogen at the bottom.

Liquid nitrogen is used pretty much everyday by someone in my lab.

Liquid nitrogen is an extremely cold liquid coming in at close to -200°C (-330°F).

Nitrogen’s natural phase is a gas. Its a fairly common gas to, making up 78% of the Earth’s air.

When it nitrogen is condensed as a liquid it is essentially always at boiling temperature.

I tried to capture the vapor coming off the bubbling liquid nitrogen in the picture above, but its difficult to convey what liquid nitrogen is like in a photo.

Liquid nitrogen looks exactly like boiling water. If you put liquid nitrogen into a pot it would look just like a boiling pot of water ready for spaghetti to be added.

But liquid nitrogen is not boiling water. It won’t scald your hand if you touch it.

Liquid nitrogen is the coldest thing you will ever touch and can instantly freeze burn your hand.

Even things that come out of liquid nitrogen are painful to touch with you hands. I can’t do it for more than a second.

Using gloves to handle liquid nitrogen has another problem attached to it.

When you wear gloves a natural layer of sweat and oil occurs between your hand and the inside of the glove.

If your gloved hand is in the liquid nitrogen for too long, the sweat freezes.

That’s just ice though. It’s happened to me plenty of times. I just yank my hand out of the nitrogen and my bodyheat melts the ice back into sweat right away.

So if its so dangerous, why do we use it in the lab?

Liquid nitrogen is useful because it stops all biological activity. That’s why its dangerous and why its useful at the same time.

When working with a dead specimen its best to prevent bacterial decay. Bacteria can’t survive at liquid nitrogen temperatures, so its used for that.

Liquid nitrogen is also used to isolate RNA from a specimen.

Every cell has RNA inside of it, but RNA is also what many viruses are made out of.

Cells quickly learn to distinguish RNA inside the cell as good and RNA outside of the cell as bad virus RNA.

Cells have defense mechanisms to destroy RNA called RNases.

RNases can’t work at liquid nitrogen temperatures though!

I was using liquid nitrogen for a third purpose today, just to quickly freeze some worms.

More on why I need to freeze worms another day!

-Mister Ed