The Story of Green Fluorescent Protein

This is a special guest blog article by Joel Lusk, a third-year PhD student in Biological Design at Arizona State University and a member of the Science on Main group. His research involves the study of photoacoustic contrast agents in the imaging and detection of cells. He can be reached at jlusk@asu.edu.

Have you ever seen the stories in the news where scientists make plants or animals glow? These stories are fascinating and amazing, and scientists accomplish these feats by using a protein that was originally found in a jellyfish! This protein is called Green Fluorescent Protein (GFP for short), and the story of its discovery and the scientists that originally pioneered its use is an interesting one. The protein itself has been used in science in a wide variety of applications to study plants, biological processes, and even the things that make up our cells.

Two mice expressing enhanced GFP with one non-GFP mouse in the
center. Source: Moen et al., 2012, BMC Cancer 12.1: 21.

 In 1960, a scientist named Osamu Shimomura started studying the bioluminescence of the jellyfish species Aequorea victoria. When the rings of these jellyfish are squeezed, they produce a glowing liquid. Shimomura claimed to have squeezed over a million jellyfish to collect this liquid and study it for his research. In 1962, he and his colleagues was the first to identify a protein in this jellyfish that he named aequorin. The aequorin first emits blue light, which is then absorbed by the GFP, causing it to glow.

The Aequorea victoria jellyfish. Copyright: Sierra Blakely.

Now that the protein that was responsible for the jellyfish's glow was identified, several other scientists became interested in cloning the gene responsible for it. If the gene for the fluorescent protein could be cloned and inserted into other organisms, it could revolutionize biology. In 1992 a biologist named Douglas Prasher suggested that this glowing protein could trace the way proteins moved in cells and was the first to clone the GFP gene. Unfortunately, the funding for his lab ran out and he left academia. Before he left, Prasher sent several samples of GFP to other scientists so they could continue his work.

After the genetic sequence was found for this glowing protein and it had been successfully cloned, the doors opened to a world of new possibilities for GFP. Prasher had predicted that this protein could be used as a reporter molecule in cells. This means that when a scientist wanted to know if a cell was producing a protein, they could just add the gene for GFP next to the gene for the other protein into the genetic code. When the cell produced that protein, it would also produce GFP, causing the cell to glow. This allowed scientists to undertake all sorts of amazing experiments, including the work of Martin Chalfie, who produced these proteins in E. coli bacteria and worms called C. elegans.

Neurons imaged from a "Brainbow" mouse. Source: Smith
(2007), Opinion article in Neurobiology 17.5: 601–608.

Another scientist, Roger Tsien, took the GFP and augmented it to be many different colors. He did this by changing the shape of the protein so it absorbed and emitted light at different wavelengths. This allowed scientists to not just label cells and animals with a green protein, but with an entire rainbow of colors! This has been applied to every part of the field of biology, allowing scientists to understand and see processes they never could before. Shimomura, Chalfie, and Tsien all shared the 2008 Nobel Prize in Chemistry for their work with GFP. Unfortunately, Prasher, the man who first cloned the protein, was not awarded the prize as three people maximum can share the prize. To honor him, all three recipients mentioned him in their acceptance speeches as even invited Prasher to the ceremony.

So now we know the history of GFP, but what are some of the cool things that scientists can do with it? Mainly scientists have used the protein to tag and visualize processes in cells. One amazing application of GFP and other fluorescent proteins was the production of a "Brainbow" mouse. This mouse produces a variety of colors in its neurons, allowing scientists to see the way the neurons interact with each other. Not only does this tell scientists a lot about how the brain works, it also creates a beautiful image!

The story of GFP is one that shows the usefulness of a fundamental science. Who would have thought that a protein first found in a jellyfish would go on to revolutionize the way we understand and see cells? Research into fluorescent proteins is still going strong, and scientists are currently looking at new fluorescent proteins that have been isolated from other sources such as bacteria and coral. Further work with the fluorescent proteins might reveal even more about the inner workings of our cells and our bodies. In the meantime, we can have a little fun with these proteins, and even paint pictures using bacteria who produce these fluorescent proteins across the visible light spectrum!

A picture painted on an agar plate with bacterial colonies expressing a
variety of fluorescent proteins. Source: Tsien Lab. Artwork: Nathan Shaner.
Photographer: Paul Steinbach.

Sources:

https://www.conncoll.edu/ccacad/zimmer/GFP-ww/shimomura.html

Chalfie, Martin, et al. "Green fluorescent protein as a marker for gene expression." Science 263.5148 (1994): 802-805.

Moen, Ingrid, et al. "Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation." BMC cancer 12.1 (2012): 21

Tsien, Roger Y. "Constructing and exploiting the fluorescent protein paintbox (Nobel Lecture)." Angewandte Chemie International Edition 48.31 (2009): 5612-5626.

Smith, Stephen J. "Circuit reconstruction tools today." Current opinion in neurobiology 17.5 (2007): 601-608.

http://www.tsienlab.ucsd.edu/Images.htm

"What's your Day Like?"

This blog article was written by Dr. Christina Forbes, co-founder of Science on Main. She earned her PhD in chemistry and is currently a post-doctoral researcher at Arizona State University. 

I was recently matched through Skype a Scientist with a 6th grade classroom at St. Hubert Catholic School in Minnesota. Some 40 students and their teacher got a chance to Skype with me while I was in my lab in Tempe, Arizona. I showed them around the lab a bit, but we spent most of the time just chatting about my work.

I met with 3 different class periods, and all three classes asked me a question that is surprisingly hard to answer: "What's your day like?" Since it was one of the burning-est questions these students had for me, I wanted to bring it up to Science on Main. After all, our aim with Science on Main is to answer your questions!

This question sometimes follows the classic "What do you do?" that comes up in polite conversation. I've learned that the attitude towards my accurate-but-short answer, "chemist," can range from excitement to disgust. Some people think that's cool, others think that sounds pretentious, and even others seem uncomfortable or unsettled with my answer.

Only the very curious, or confident, have asked me the follow-up: "So what's your day like?" 

Indeed, this is just my job, and it's a job I like and I worked hard to get to, but it doesn't endow me with any particular superpowers. I don't know how much people in other professions are asked this question, but I hope everyone is. There is so much fascinating stuff that can go into one person's workday. What does a software engineer do on a day-to-day basis? Or an investment banker? Or an event coordinator?

Nonetheless, these 6th graders were very curious. If you are also very curious, then I can give you a detailed approximation of my answer to "What's your Day Like?"

[Get on with it!]

On an average day, I don't function well for the first hour, so I sit at my laptop with my coffee before I attempt any chemistry. Yup, even scientists have morning commutes and laptops. I deal with emails, follow up on other emails, check my calendar for scheduled meetings, update my calendar based on the emails I got that morning, and other desk-stuff that I have to do to keep my life in order. I might edit a peer's manuscript for a paper they want to publish, or I might finish writing a draft of something that I'm working on. I might catch up on reading recent literature, like reading the news but it's only the chemistry section, written with all the jargon. When the coffee has kicked in and I think I'm a little more ready to face my day, then I'll pull out my lab notebook, put on safety glasses, and go into the lab.

My day in lab will focus on a different part of a project that I might be working on. To save chemistry jargon, I will use an analogy for my project: making a batch of THE BEST cookies for a friend. Let's say I chose, with some reasoning, to make chocolate chip cookies for my friend.

Troubleshooting a reaction:
I'll need a good recipe for the cookies. I probably looked up recipes online, maybe chose a few recipes based on reviews or ingredients that I have on hand. Maybe I asked a buddy in another lab what recipe they use, or if they have feedback on a recipe they tried. Once I have a recipe I feel good about, I do a test reaction, or a small batch. I look at the product (in this case, the cookies) to see if I could have done the reaction differently, or if this recipe is terrible and I need to try a completely different one. I'll do a series of these test reactions until I obtain the cookies I want, then I scale up the reaction and try a larger batch. Sometimes I'll have to adjust the recipe a bit more when I make a larger batch, like a longer baking time or less salt. All the while, I make notes on how I set up each batch of cookies, and how the cookies came out. What was my baking time? Baking temperature? How much sugar? Should I use honey instead of sugar? Were the cookies undercooked? Burned? Too salty? I might spend a month or more testing and perfecting a recipe for cookies in these batches.
For those of you trying this at home, this might look like running reactions, refluxing reactions, distilling materials, running a purification column, mixing chemicals, etc.

Synthesizing a molecule I need:
In the midst of testing my cookie recipe, I run out of chocolate chips. Nope, the store doesn't sell any chocolate chips, but the store does have a lot of cheap cocoa beans. After a little more internet searching, I learn how to make chocolate chips from the beans, and find that I have the equipment to roast, winnow, grind, and, generally, make chocolate. Since I've never done this before, I may have to borrow some techniques I know about about making coffee from the beans, or borrow equipment from a neighboring group. Much like the cookies, I practice making the chocolate from the beans in small batches, but it comes out gritty and yucky. I work on refining my chocolate to get the grittiness out before I can make it into chips. Once I have a few chocolate chips that are right for putting into cookies, I'll make a bigger batch so that I have plenty to use for testing out my cookie recipes. All the while, I've made notes on each step of the process to make the chocolate. That way, I've built a recipe that I can follow if I need more chocolate chips.
This might look a lot like troubleshooting reactions. I bring this up because sometimes there are materials that I can't buy, and I have to take a step back in my project to make what I need for myself. So much of this stuff is self-taught.

Something as simple as removing liquid from a solution requires this contraption.

Drying a purified product under vacuum.
Imagine chocolate chips, I guess.

Characterizing:
Remember, I'm trying to make these cookies so that my friend thinks they're THE BEST. To do that, my friend will need some other cookies to compare against mine (think control experiments). Maybe they have another friend with jerk chocolate chip cookies, and another friend that made some ginger snaps. After trying all these different sample cookies, my friend thinks that mine are THE BEST, but we should probably check again another day, just to be sure. More cookies are made, and each sample cookie is brought to the friend the next day, and they still think mine are THE BEST. But, there's a rule of three in replicating results, so we should check again next Monday.
I'm using a person and their sense of taste as an analogy for taking a measurement for making comparisons.
This is where the edge of science really happens, and it can depend on the project. It could be taking a spectroscopic measurement, or it could be comparing different reaction rates. This is often in front of a computer that it attached to some very expensive instrumentation. Knowing if your method or product is THE BEST is revealed by a number that the instrument tells you. It could be a simple as taking a measurement of a sample, and the number 550 means that a month's worth of tireless hours of work has paid off. It's very exciting, but it probably doesn't look as exciting as other parts of my work.

Sample preparation for a spectroscopic measurement.

Measuring pH in a series of samples. UV-Vis spectroscopy came a little later.

Squiggly-lines and peaks coming from instruments is sometimes the most exciting part.

Fixing equipment:
Naturally, the following Monday, my friend has the flu, so I spend every day for a week with my friend. I need to get them back to good health so they can try all the cookies a third time. When my friend tries the cookies a third time, they think those other jerk chocolate chip cookies are THE BEST. This seems weird, since it's not consistent with our earlier tests. I have my friend try the sample cookies the next day, for a fourth time. My friend now likes ginger snaps, and they seem very confused and disoriented. I might spend a few days studying human anatomy, and realize that my friend probably has a blood sugar problem, and now it's time to call a doctor. Since doctors can be really expensive, my boss asks me to correct my friend's blood sugar problem myself. I might have to study human anatomy for two more days, only to realize that my friend is having a calcium deficiency, which effects their ability to taste anything. I give them milk, and my friend is immediately feeling better. My friend tries the cookie samples, and mine are THE BEST. For good measure, I ask my friend to try the cookie samples three additional times over three different days, just to be extra sure of the result.
A frustrating part of my job is when I've had to spend whole days away from my projects just fixing instrumentation. We need our instruments to be accurate, well calibrated, and well maintained. In my lab, these instruments include spectrometers, chromatographs (gas or liquid), or potentiostats. A service technician can be expensive for on-site service (e.g. house call), so we often learn how to do repairs and maintenance ourselves. These repairs can range from cleaning sample holders to needing replacement parts that you can only describe with picture messages.

That time we had a problem with a part that isn't normally part of the wear&tear kit and doesn't have a part number... 20 emails, 3 manuals, and many pictures later, I managed to communicate to the service tech that I needed a new needle port tube.

Because a degree in chemistry sometimes means that you've had to figure out how to disassemble, diagnose, and service an injector for an HPLC. (Really, what does that even mean?!?)

The tube had been mangled--not sure how, or how long it was like this.
The guide did not fit on the old tube, thereby ruining the fluid seal, thereby disrupting sample injection for analysis, and grossly ruining the data.
A reminder that scientist does more than just experiments and mixing chemicals!

Publishing:
Since my cookies are THE BEST, now I need to publish my recipe. I write out my recipe, with pictures of what they look like and a picture of my friend eating each sample cookie (they're happy only when they're eating my cookies). I attach a supplemental document that describes how I made the chocolate chips from cocoa beans, with some information about my friend who tried and judged the cookies. I send all of this to a bakery owner, and they send my recipe to other bakers. These other bakers submit feedback about my recipe, asking me to have my friend try some other cookie types. These other bakers also think that my cookie baking time is a little weird. I spend some time doing what the bakers ask, subjecting my friend to more cookies, and refining the cookie-making process. I send a revised recipe and more photos of my friend eating all these other cookies, and the bakery owner agrees to publish my recipe in their next book.
This is a very broad brush-stroke of the peer-review process. This is often a background think to my work, but dealing with reviews and doing new experiments can become part of my average day.

Copyright prevents me from posting my papers, so instead you get a picture of my dissertation.
For the record, a doctoral dissertation should not be this long.

Training new colleagues
While I'm working away at perfecting my cookie recipes, my boss walks in with a college student and says "this new student wants to learn how to make cookies. By the way, they've never been in a kitchen before, but it would be great if they had a batch of cookies by the end of the week." My boss leaves, and the student looks at me with excitement and fear. We may as well start with the batch I'm working on, and I show the student how to use a spoon to stir the dough. I step out of the kitchen for a moment, and when I return, I find the dough on the floor. Maybe this new student was stirring the batch too fast and lost control of the spoon and the bowl. I tell the student that it's ok, but now they'll have to help me start over. It's a part of the learning experience, and if they pay attention, they'll get better with time.
As I explain the recipe and how to stir the dough, the student seems to think I know everything about cookies. I explain that I wasn't born with cookie knowledge, but I just worked with cookies for a long time. I tell them that if they work with cookies long enough, and learn from their own mistakes, then they'll know a lot about cookies, too.
As you gain more experience in your project or techniques, you're expected to help train new people. You learn a lot of patience when you train new people, but it's crucial to the learning process. I sure know that I tested people's patience when I was still learning this stuff! 


Each day in lab can be very different, since each day might be part of this overall process of making and testing THE BEST cookies. At some point during the day, I eat lunch (maybe). Sometimes I might meet briefly with my boss, and we talk about how cookie-making is going. Maybe not; I've earned some independence with cookie-making.

By the end of the day, and when I've brought my work to a natural close, I catch up on my emails or whatever else happened at my desk while I was in lab. I write out a new to-do list for the next day, hopefully with notes to myself like "make another lb. of chips" or "try the recipe on page 42 with more butter." I close up my laptop and go home. I try throw together dinner. Maybe I look at a colleague's manuscript while my husband works on his own homework, or maybe I work up and analyze some new data that I can't wait to see the next day. Or maybe I'll just read a book or watch Netflix. Scientists just need to chill sometimes, too.