Author Archives: Erin Brooks

“Find your calling,” Dr. Lefkowitz advised us when he spoke to the Howard Hughes Research Fellows this past Tuesday morning. Whether its in music, math, or oceanography, find that calling and follow it.

If a Nobel laureate ever gives you advise, you should probably take it. But how does one find their calling? How do you know for sure that you are destined for a life of research instead of, say,  medicine or, I don’t know, competitive fly fishing? Even Dr. Lefkowitz, himself, admitted that eight weeks in a lab is not nearly long enough to give you a true taste of what a life of research entails. In fact, he described to us how he hated his first year of research at the NIH so much that he vowed never to go into research. Dr. Lefkowitz originally believed that his calling was to become a physician. Nearly forty years and one Nobel prize later it turns out that he couldn’t have been more wrong, solid proof that it takes time to find your true calling.

So how has my eight week experience affected my career choice? Honestly I have no idea. Its too early in the game to say for sure. What I do know is that I don’t intend for this to be the end of the road. Eight weeks was long enough only to grant me a teeny, tiny taste of the research life, but it wasn’t nearly long enough for me to decide whether I’m suited to commit my life to it. Nevertheless, I just might be hooked. But, like I said, its too early to say for sure. And what’s the rush anyway? Isn’t that what being an undergrad is all about? Trying out new things, pursuing weird interests?

The way I see it, the best way to find your calling is to start out with a list of all your interests and then start crossing things off that list. This summer was just the beginning of that process. And I discovered research is definitely something I don’t want to cross off my list just yet.

So what happens if I continue on in research? How does that work? Listening to the weekly seminars given by Duke faculty has given me a better understanding of how the career of a scientist pans out. Through the seminars, I’ve also realized that there is no single set in stone path to becoming a researcher. Several of the faculty who spoke to us confessed to originally wanting to become doctors (because who doesn’t want to become a doctor, right?). Several others said that research just found them in a serendipitous moment of new discovery. But no one said that he or she just simply knew from day one that he or she wanted to go into research. I guess the point is that no one knows for certain whether or not he or she is cut out for a life of research. All you can say for certain is that you are passionate about what you are studying. The biggest take-home message: be passionate about your research.

Susan Alberts’ faculty seminar, in particular, stood out to me. Dr. Alberts works in the Duke Department of Biology studying the social behaviors of Amboseli Baboons in Kenya. For part of the year, every year, Dr. Alberts settles down in a tented camp at the base of Mt. Kilimanjaro in Kenya to monitor the social interactions of the population of Amboseli Baboons that live there.

Dr. Alberts described three key areas of interest concerning the baboons’ social interactions: social relationships, social status, and parental care. These three research interests, taken together, help to define how each individual baboon survives within the larger context of the family or group unit. Dr. Alberts seeks to answer several questions. How does social isolation put female baboons at greater risk for death? How does social rank affect the nutrition and relationships of females? Do male baboons invest preferentially in their own offspring? These questions are compelling on their own, but they also seem to have echoing implications for studies into human behaviors and social interactions as well. For example, the fact that social isolation puts baboons at greater risk of death than socially integrated baboons seems to also be true for humans as well, see here and here.

In addition to describing her research interests and discoveries, Dr. Alberts also gave us some insight into what her life was like living in a tent at the base of Mt. Kilimanjaro without the luxury of modern day conveniences. I enjoyed this aspect of the talk as much as I enjoyed learning about her work. Research isn’t just a job, its a lifestyle. This is the major lesson I gleaned from Dr. Alberts’ talk. A life as a researcher is an all-encompassing one, but it is also a truly enjoyable one when you are pursuing a question or idea that you are truly passionate about. Dr. Alberts’ work is the perfect example of how pure passion and dogged interest can generate fruitful and compelling scientific results.

Full disclosure: no matter how hard you try, no matter how careful you are, things can, and most certainly will go wrong. I say this from first-hand experience. Allow me to explain. As I described in my previous post, I am working with chromobodies, which are tiny nanobodies fused to a fluorescent protein. To create our chromobodies we are using a new procedure known as gateway cloning. You can read up on the procedure in more detail here, but basically the process involves two steps in which the DNA sequence for our chromobodies is inserted first into one plasmid and then recombined into a second plasmid to create an expression clone. The expression clone is a plasmid containing a promoter that will cause the chromobody to be overexpressed within the cell. The idea is that once we transfect cells with our expression vector, they should begin producing chromobodies that will bind to their antigen targets (either actin, lamin, pcna, or dnmt1) within the cell. We then put the cells under the confocal microscope which excites the fluorescent proteins within our chromobodies allowing us to visualize the chromobodies bound to their targets within the cell. Here’s a time-lapse video of cells that have been transfected with a lamin chromobody containing GFP (green fluorescent protein):

I have finished cloning the DNA sequences for each of my four chromobodies into four different expression vectors (using the gateway cloning protocol).  And I have also transfected HEK cells with my four different chromobodies.

So far so good right? Except things happen, things you can’t control. For example, you might get only three out of your four chromobodies to successfully transfect. This is what happened to me. Our chromobody for pcna would not transfect. We sequenced the DNA for our chromobody and low and behold discovered that it contained a mutation that caused the fluorescent protein to be dysfunctional (meaning that it did not emit light after being excited by the laser in the confocal). So what do you do? You go through the whole cloning procedure again and hope that the next time around you don’t get a mutation. Of course, as luck would have it, my second clone also had a mutation. It was a different mutation, but it resulted in the same loss of chromobody functionality. So right now we are currently on round three of cloning for our pcna chromobody.

Moral of the story: when life gives you a mutation, clone, clone again.

While listening to my fellow peers give their chalk talks this week I was struck by how different everyone’s research projects are from one another. Some people are working in Matlab, others are amputating fish fins or watching plant seeds germinate, and still others are analyzing the structure of mantis shrimp claws. Our research projects truly run the gamut, from cell biology to plant biology to computer science. All of the sudden it hit me: research can take on a variety of forms. Ok, so this probably shouldn’t come as a big surprise, but I guess I had just assumed everyone was doing basically the same things that I’ve been doing: cell culture, PCR, gel electrophoresis, DNA sequencing. While its true that some people are doing these things as well, its also true that most people’s daily research routines are far different from mine.

For example, I want to focus on Matt’s research, which I found particularly interesting as well as completely different from my own research. Matt is working in Matlab to create a method for quantifying the pain that patients report having before and after treatment. I think his research is so interesting because it tackles such a tricky and difficult question. How exactly do you quantify pain if the sensation of pain is a subjective experience, unique to the individual? Patients suffering from the same disease may report experiencing pain in different areas of the body. These patients may also report different levels of pain in those areas. So how does the health care provider determine whether their treatment is working for a particular patient? This is the problem Matt’s research is designed to solve.

Before treatment a patient is given a sheet of paper with the outline of a human body printed on it. The patient then shades in the region of the body in which she is experiencing pain and rates the level of her pain on a scale from one to ten. It is Matt’s job to convert these visual representations of a patient’s pain into concrete data. It seems like a simple task until you consider the fact that Matt is working with hundreds of these pain diagrams, all of which must be scanned and ‘translated’ into a language the computer can understand.

As Matt explained the steps he has to take to make the diagrams ‘understandable’ to a computer, I began to understand how difficult and frustrating the process must be. First he has to crop the diagram so that only the shaded regions within the body are registered as areas with pain (sometimes the patient scribbles outside of the lines, which we assume does not mean that the patient is experiencing pain outside of his or her body). Next Matt has to fill in the shaded area completely (eliminating the white spaces within a shaded area so that the entire area is a solid color). Once the area in which the patient reports having pain has been properly defined the computer can then measure what percentage of the entire body is affected by pain. After treatment the patient will be asked to fill out another diagram to report his or her pain. Then the area of pain the patient reported having before treatment will be compared to the area of remaining pain reported after treatment.

I can only imagine that the work that Matt is doing in Matlab to accomplish his intended goal of quantifying a patient’s reported pain is difficult and at times extremely frustrating, especially since he has never worked in Matlab before. It must be easy to feel overwhelmed. I admire his perseverance (Matlab is notoriously hard to learn) and I’m really impressed by how much he’s been able to accomplish in only a month’s worth of work!

So what exactly is my typical day like?

Well, I’d like to tell you that I arrive at the lab each morning with my entire day planned out. My imaginary self slips on her gloves and immediately gets to work cloning her geneblocks, transforming bacteria, running gels, or transfecting HEK293 cells. She knows exactly what refrigerator to look in for that one primer that her mentor told her to find (and doesn’t need to look through all of the how ever many refrigerators there are in the lab, twice). She doesn’t forget to turn the power on when she runs her gel, nor does she need help running a centrifuge or spectrophotometer because both of those gadgets are just so darn easy to operate. Oh, and also, did I mention that my imaginary self has perfect coordination? Never spills anything. Nothing. Not once. Never.

You see, my imaginary self has been working in the lab for four weeks and naturally has it all figured out by now. To be sure, four weeks is certainly enough time for anyone to master the techniques required to work in a lab. I mean that’s a whole month of working over forty hours a week. That’s 160 hours, or 9,600 minutes. The point is: don’t you think that’s a lot of time? So one would expect that the real me would not be making any mistakes. The real me shouldn’t be screwing up her gene cloning by adding an enzyme too late. She shouldn’t accidentally plate too many cells causing them to be confluent the next day. She shouldn’t spill, splash, break, or drop anything. Right?

This is what I tell myself every day when I arrive at the lab. I tell myself “NO MISTAKES,” and then, without fail, I screw up. This is the only consistent thing that occurs from day-to-day in my lab. I may not know what exactly I’ll be doing when I arrive in the lab each day, but I do know I will goof up at least once. Its only a small comfort that my number of screw-ups per day is down at least 50% from my first day. I’m not complaining. I’m actually amazed at how far I’ve come in these past four weeks. It’s like my mind is expanding like a balloon. My eyes are constantly popping out of my head. I may have a daily mistake, but I also have a daily eureka moment when I discover something new (something new to me at least), or have a sudden insight into some cellular process. It’s those eureka moments that keep me coming back to the lab day after day.

Cellular biologists are generally concerned with four major cellular processes: mitosis, apoptosis, DNA methylation, and DNA replication. The aim of my project is to design a tool to allow researchers to interrogate these four major cellular processes in a living cell. Such a tool will have valuable implications for research on a wide range of diseases from cancer to Parkinson’s. 

The basis for this tool is a special type of tiny antibody found in camelids (alpacas, camels, llamas, etc.), known as the nanobody.  We are working with nanobodies that bind to four different antigens endogenous to the cell: actin, lamin, proliferating cell nuclear antigen (PCNA), and Dnmt1. Each nanobody is also fused to one of four different fluorescent proteins: mTFP (teal), mKO2 (orange), EYFP (yellow), and tagBFP (blue). By fusing a different fluorescent protein to each of our four nanobodies, we are able to image (via a confocal microscope) these antibodies as they bind to their target antigens within the cell.

The logical question here is “So what?” Of course, its cool to look at a cell that’s glowing four different colors, but why is this important to science? Well, as I mentioned in my intro to this post, apoptosis, mitosis, DNA methylation, and DNA replication are the four major cellular processes that cellular biologists are concerned about. As it turns out, each of our fluorescently tagged nanobodies, which I will now refer to as chromobodies, tell us something about each of the four major cellular processes. Our actin chromobody binds with actin, a major component of the cell’s cytoskeleton, thus allowing us to characterize the cell’s shape and structure. Our lamin chromobody binds with lamin, a component of the nuclear lamina which encloses the cell nucleus, allowing us to visualize the cell’s  nucleus. Dnmt1 is responsible for DNA methylation, therefore our Dnmt1 chromobody allows us to see where transcription of the cell’s genome is being turned off due to methylation. Finally PCNA is a molecule that must be present for successful DNA replication, so our PCNA chromobody allows us to see when a cell is replicating its genome.

Thus, our four chromobodies can effectively act as a toolkit allowing us to interrogate a cell about its activities. Kind of neat, right? Right now we are creating plasmids containing the sequences of our chromobodies (via Gateway Cloning) and then transfecting cells with them. Once we have live cells expressing our chromobodies, we can look at look at these cells with a confocal microscope and ‘ask’ them a series of questions. Is this cell getting ready to die? Is this cell getting ready to divide? What are the patterns of DNA methylation? The answers to these questions are particularly pertinent to the Caron Lab’s main research goals. These goals include better understanding how G-protein coupled receptors (GPCRs) mediate the development of disease.

And this is only the beginning. The next step will be to take this technology and apply it to cancer research. The long term goal is to fuse four different fluorescent proteins to a wild-type oncogene and three of its mutations. We can determine which of the four genes are randomly expressed (in a mouse model) based on the coloring of the cells. From there we can compare cells within the same animal model to see the varying effects of different oncogene mutations on the fate of a cell. Note that this description of the lab’s long-term research goal is a basic, shallow summary. Right now I will not go into depth about the methods and mechanisms required for such a project. Here I am merely giving a general, plainly-worded summary of our goals. Just three weeks ago I had no clue this kind of thing could even be possible. I find it thrilling that I’m getting to play even the tiniest role in this larger body of exciting new research.

My primary mentor, Dr. Marc Caron, was out of town this past week, so I interviewed the lab PI, Dr. Larry Barak, instead. Dr. Barak has been working in the Caron lab for almost 23 years now. He majored in physics and math at the University of Michigan, then went on to Cornell where he got his PhD. He later returned to the University of Michigan to get his MD. After ten years of working as a pediatrician, he decided that he wanted to get back into research. Through a fellowship program funded by the Howard Hughes Medical Institute, he was able to find a job at the Caron Lab where he has been working ever since.

Curious about how Dr. Barak first became interested in research, I asked him about his first research experience. As an undergraduate at the University of Michigan, he had a work study job potting electrode tubes at the National Accelerator Laboratory. Although the job was fairly routine, it caused him to lean towards research as a career.

Dr. Barak’s research focuses primarily on G-protein coupled receptors (GPCRs) and their implication in addiction. He does this research in conjunction with the National Institute on Drug Abuse (NIDA) which funds his research. I asked him if his academic background as a math and physics major helped him to approach scientific problems from a different perspective. Surrounded by molecular biologists and biochemists, he finds that this is often the case. He told me that the best way to solve a complex scientific problem is to engage it through multiple academic disciplines. For this reason, he says he takes a team based approach to problem solving.  When I inquired about the steps he takes when trying to answer a scientific question in the lab, he offered me a bit of sage advise, “you have to participate in the problem before you can solve the problem.” If you can’t fully articulate the question, then you won’t know where to start looking for the solution.

Having spent nearly 23 years in the Caron lab, I asked Dr. Barak about how things have changed since he first arrived. Unsurprisingly, many of the procedures and pieces of lab equipment that were routinely used when he first arrived in 1992 are now outmoded. He described to me how much of his work is moving towards interrogating cellular processes in real time via confocal microscopy. He told me about the new ability of researchers to use fluorescent proteins to view cellular processes in live cells. In relation to Dr. Barak’s work with NIDA, this new method of imaging cells provides researchers with a better way of looking at the effects of certain drugs on specific cellular processes.

I asked Dr. Barak if there was anything he would like to change about the world of research. He feels that the US needs to change the way it funds research. Repeatedly having to apply for grants is a significant drain on the valuable time of a researcher. Although, on the other hand, guaranteed funding isn’t necessarily ideal either. It doesn’t promote creativity or supply sufficient pressure on researchers to engage important research questions. We need a funding system that is somewhere in the intermediate, Dr. Barak told me.

Before tying up the interview, I asked Dr. Barak if he had any funny stories to tell from his 23 years in the Caron lab. He couldn’t think of any fires or major catastrophes, but he did recall one time when an unnamed someone left the burner on under a coffee pot resulting in so much smoke that the lab had to be evacuated. The event was quite ironic. Apparently scientists don’t make the best coffee after all.