Author Archives: Caroline Gamard

So long, farewell!

You really can’t imagine what research is like until you actually do it.  Coming into this summer, I had absolutely no idea what working in a lab would entail.  When my friends and family asked me how I would be spending the summer, I answered that I knew I was working in a biochemistry lab, which was about the extent of my knowledge about what I would be doing.  I obviously didn’t know what I was getting myself into!

That being said, I am so grateful to have had this opportunity this summer!  Transitioning to lab work can be pretty difficult, overwhelming, and scary, but BSURF provided great advising and mentoring and most importantly, a community of other students who were going through the same transition.  I have really loved getting to know everyone, and I hope I have formed connections that will last throughout my time at Duke and beyond.

I have learned a lot about the nature of research and lab work this summer.  First of all, eight weeks is not a lot of time.  By the time I really got a grasp on what I was doing, it was almost time to leave.  I need to devote a lot more time to a project to get rewarding results.  Second, stuff goes wrong.  In fact, sometimes I think it goes wrong more often than it goes right.  There is always some setback or unexpected problem.  Data doesn’t look nice.  I forgot to add ATP to a sample, or I don’t have enough protein to do an important assay.  A machine gives me a crazy error message the one time I’m completely alone in lab with no one to ask for help.  In the words of my mentor, “that’s science.”  Research is not like the lab component of my chemistry class.  It is messy and confusing, and sometimes you have no idea what’s going wrong or right.  However, my final discovery was that research can be incredibly rewarding.  The first time I quantified a protein gel, put the data in Excel, and created a graph that strongly indicated the expected result, I was amazed.  Hours of work had gone into the creation of that data set, and it was an actual result that I could potentially use.  There is really nothing like the feeling of accomplishment that I felt generating that graph or the feeling of seeing my name and results on a 42 x 36 poster.

I am beyond grateful for this experience: this summer was really the perfect time for me to test out the world of research without having to balance the demands of the academic year.  I can’t wait to see where the future takes me, but I am even more excited to keep up with the scientific accomplishments of the friends I have made this summer.  As Dr. G says, science is communication, and I know that the truly amazing people I have met here will have some awesome research to communicate soon!

So goodbye BSURF and thank you!

Stories of incredible strength: mantis shrimp, trap-jaw ants, and Dr. Sheila Patek

I was struck by Dr. Sheila Patek’s talk about her scientific journey, particularly because she faced a completely unexpected challenge that wasn’t something I previously would have considered a roadblock for scientists: politics.  Dr. Patek’s research almost lost funding when it was identified by a senator as something that was an unnecessary use of taxpayer dollars.  Dr. Patek researches extreme movement in animals such as mantis shrimp and trap-jaw ants, which admittedly does sound like something that is not immediately necessary to move the field of science forward.  However, plenty of scientific research seems to be based on learning more about the world for the sake of learning more about the world, not learning for the sake of advancing technology or saving humanity.  Isn’t learning for the sake of learning what science is all about?  That’s what I thought before Dr. Patek told us about how her research was challenged for being unnecessary and how she was told that it was not worth the time or money that she poured into it.  I never considered that this would be a challenge for a researcher, someone who spends his/her life learning about the world.

Dr. Patek’s talk taught me three important lessons that I will remember as I grow as a scientist:

  1. Learning more about the world for the sake of learning more about the world is of the utmost importance. We should study everything, however seemingly insignificant and try to gain as great an understanding of our world as we can.  That’s our job as scientists.  If something gives me a greater understanding of some aspect of the world, it’s worth doing, even if I can’t immediately see the applications or the way that it advances society.  It doesn’t have to solve a human problem to be important.  The study of how mantis shrimp resolve conflict is just as important as the study of neuroscience, cancer biology, or evolution.  Just because one has more obvious implications for humans does not make it more important.
  2. That being said, all research has implications for humans. Although Dr. Patek’s research was challenged for its lack of relevance, she proved that it does have really important applications in engineering and technology and our understandings of evolution and physics.  Although these applications are not immediately obvious, they still exist and are of great importance.
  3. It’s important to stand up for ourselves and what we love and believe. When Dr. Patek was challenged, she did not sit idly by and lose funding.  She met with the senator on Capitol Hill to convince him that her research deserved funding, and she also spoke about the importance of research on PBS.  Dr. Patek’s bravery and perseverance in the face of adversity are truly inspiring to me, and the lesson I learned from her actions about standing up for knowledge and standing your ground is extremely relevant inside and outside of lab.

I highly recommend everyone watch Dr. Patek’s PBS Newshour segment.  She speaks so eloquently about why research is important for its own sake, and it’s a very inspiring message, especially for us as the next generation of researchers:

Abstract thinking

Inhibition of HipA to Reduce Multidrug Tolerance in E. coli

The HipBA operon is a bacterial toxin-antitoxin module that plays a crucial role in multidrug tolerance in E. coli.  HipA, the toxin, is a kinase that functions by phosphorylating translation factors, inhibiting translation in the cell and inducing a state of dormancy in which cellular processes that are targeted by antibiotics are shut down, allowing the cell to evade antibiotic poisoning.  The goal of my research is to identify molecules that bind to HipA and inhibit its kinase activity.  HipA autophosphorylates, so its activity can be measured by its phosphorylation.  To measure phosphorylation, wild-type HipA is completely dephosphorylated using a phosphatase enzyme, and its phosphorylation after the addition of ATP can be visualized through a ProQ Diamond Phosphoprotein Gel Stain.  This assay can be repeated with wild-type HipA in the presence of target molecules that are believed to inhibit its kinase activity.  A lack of autophosphorylation in the presence of a molecule indicates that this molecule inhibits HipA and could be a potential molecule of study for the development of an antibiotic.

A day in the life

Dear Diary,

Today was a pretty typical day in lab.  I completed a phosphorylation assay using 10% DMSO to make sure that the assay works with DMSO.

I started out the day by stopping the dephosphorylation reaction that ran overnight.  The reaction mixture included dephosphorylated HipA, MnCl2, lambda-phosphatase, and buffer solution.  I began by isolating the dephosphorylated HipA using a Ni-affinity chromatography column.  After the protein was isolated, I needed to concentrate it up because a specific concentration is required for the phosphorylation assay.  I centrifuged 3 mLs of the elution fraction at a time, concentrating the protein.  Next, I needed to buffer exchange since the buffer used for the dephosphorylation reaction is different from the buffer required for the phosphorylation assay, so I centrifuged the protein three more times, diluting it by a factor of five with the new buffer each time, resulting in a 125-fold dilution.  I checked the concentration of the protein and found it to be 0.71 mg/mL, indicating approximately 70% yield, which is pretty good.  I left my protein in the fridge and headed to lunch.

After lunch, I began the phosphorylation assay.  I have already done this assay twice, but today I needed to try it with 10% DMSO to make sure that it gives the same results as without DMSO.  The compounds that I will test later are kept in 10% DMSO, so it is important to make sure that this will not affect my results and that the usual assays still work with DMSO.  I diluted my protein to a concentration of 0.05 mg/mL in assay buffer and put 500 uL of this dilution into one tube, and 450 uL of this dilution into a second tube.  I also added 50 uL of 100% DMSO to the second tube to create a concentration of 10% DMSO to mimic what the compounds to be tested later are stored in.  I removed 20 uL samples from each tube to serve as my controls.  Then I added 5 uL of ATP to each tube and incubated them at 37°C, beginning phosphorylation.  I removed 20 uL samples from each tube after five minutes, fifteen minutes, thirty minutes, forty-five minutes, an hour, and two hours.  After removing the samples, I immediately heated them at 99°C for five minutes to denature the protein and stop the reaction.  I then added loading dye and stored them in the fridge for later when I will run the gel that will show me my results.  The purpose of removing samples at different times is to demonstrate how HipA auto-phosphorylates in the presence of ATP over time.  I will compare the results of the protein with and without 10% DMSO to determine if the DMSO affected HipA’s auto-phosphorylation or if it affects the phosphostain procedure that I will use to visualize the gel.  If the two gels look the same, I am safe to use this procedure with the test compounds.  If the gels do not look the same and the DMSO does in fact affect the assay, I will have some troubleshooting to do before I can test out the compounds.

Overall, a usual, but rewarding, day in lab.  I can’t wait to stain the gels and see what my results are.



If mice could talk

I really enjoyed Evelyn’s chalk talk on the ultrasonic vocalizations of mice because it presented a fascinating solution to a question I never would have thought to ask.  Her overarching question is whether or not mice are aware of their own vocalizations.  Before hearing her talk, I never would have considered the fact that mice might not be aware of their own vocalizations or that this ability that humans have to distinguish between self-generated and foreign sounds is actually pretty special.  Furthermore, once this interesting question was posed, a very creative way to test it was generated.  I admire the way her lab came up with a unique solution to a unique question.

In a nut shell, the lab tests whether mice can distinguish between self-generated and other noises by giving them helium and seeing how/if they react to the raised pitch of their voices.  If they adjust their vocalizations to a lower pitch or lower the volume of their vocalizations, it could indicate that they can distinguish which sounds they are producing and that they can discern that they sound different than usual and adjust accordingly.

However, even more interesting than the idea of giving mice helium to see if they notice when their voice changes is the important implications that it has for humans.  I was also impressed by this aspect of the presentation because while this research seems interesting and relevant to the study of neuroscience in mice, it is not immediately clear how it applies to humans.  Evelyn explained that this research can be applied to the study of schizophrenia, as people with schizophrenia often have difficulty distinguishing between self-generated and other sounds.  Thus, research on the ultrasonic vocalizations of mice can be used to better understand a disorder that affects hundreds of thousands of people and is not currently well understood.  That’s one of the most amazing things about science and research that I’ve discovered this summer: it always has broader implications that can make our world a better place.

So thanks Evelyn for the fascinating talk!  I can’t wait to learn what you discover!

Get to know Dr. Brennan!

Dr. Richard Brennan began his research journey at Boston University.  He started as a chemistry major but switched to biology, although he maintained his interest in chemistry, which ultimately led him to the field of biochemistry.  He was also interested in history and English, and he emphasized to me the importance of the humanities in science.  Without the ability to communicate, science is meaningless, and scientists need to be good writers in order to effectively write informative and intelligible papers.  Thus, Dr. Brennan’s interest in language and English proved especially beneficial for his life of publishing papers and writing reviews.  This advice from Dr. Brennan made me appreciate the interdisciplinary curriculum I enjoy at Duke: my humanities classes are just as crucial in preparing me for a career in research as my science classes.

The summer after his sophomore year of undergrad, Dr. Brennan participated in a summer research fellowship much like BSURF during which he decided that he might like to pursue research as a career.  After graduating from Boston University, Dr. Brennan went to Cornell University for graduate school, but he quickly realized that he needed to take a break before continuing his education.  He left and took a position as a technician at a hospital in Boston.  This taught me that there isn’t a set career path to research: it’s okay and even important to take breaks and explore other options and fields.  Later, Dr. Brennan returned to school and earned his PhD at the University of Wisconsin and then went to the University of Oregon for his postdoctoral fellowship.  Dr. Brennan emphasized to me the importance of good mentorship: he took his first job at a medical school in Portland because it was geographically close to his mentor at the University of Oregon, allowing them to continue to work together.  He became a full professor at the medical school in Portland.

After seventeen years, Dr. Brennan moved to MD Anderson Cancer Center in Houston to establish a structural biology center there.  However, after six years, he was offered a job as the Chair of the Department of Biochemistry at Duke University.  Dr. Brennan emphasized how much he enjoys working with students and how he thinks graduate students are essential to laboratory research.  Accepting the position at Duke would allow him to work with highly motivated graduate students, and Duke had an excellent grad student to faculty member ratio in its biochemistry labs which would allow the Brennan lab to bring in many graduate students.  Furthermore, a position as chair of the department would allow Dr. Brennan to build a strong department at an already great school.  Needless to say, Dr. Brennan accepted the position at Duke, and the rest is history.

One takeaway from Dr. Brennan’s journey is that amazing opportunities are often unexpected: he was not looking to leave MD Anderson, but he received an amazing offer to do what he loves most at Duke.

Dr. Brennan truly loves students and teaching.  He believes graduate students are what make research labs great, and he also believes that all faculty members should teach.  Dr. Brennan teaches everything from courses on grant writing to x-rays to his specialty, structural biochemistry, and he usually has at least two undergraduate students in his lab each year.

While Dr. Brennan loves research because of the unique opportunity it gives him to observe something that has never before been observed and to understand something that has never before been understood, he offered me some warnings about the nature of the field.  He encouraged me to study topics that interest me, rather than those considered “hot” in science right now.  Brilliant research is being done everywhere, but not all of it is recognized because not all of it focuses on what is considered new and important in the field at the time.  Focus on what you love, not on what will win you awards and national recognition.

Dr. Brennan’s final advice to me was to seize all the opportunities available to me and try as many different research settings as possible until I find one in which I feel truly comfortable and happy.  There are many different research experiences, and I cannot know how I really feel about conducting research after this one experience, however great it may be.  I should never close myself to other opportunities just because I am comfortable where I am: Dr. Brennan loved his job at MD Anderson, but he was open to a change and got to create an amazing department at Duke, where he is very happy.

TL;DR: Always be on the lookout for new opportunities and never stop pursuing what interests you!

Facing my fears

While many people (not Dr. G) fear snakes or spiders, I have been afraid of antibiotic resistant bacteria since I read a book about it in middle school.  But unlike my other fears, I don’t want to avoid antibiotic resistant bacteria.  I want to do something about it.  That’s one of the reasons why the Brennan lab stood out as a good match for me this summer.

My project in the Brennan lab is primarily focused on a bacterial toxin called HipA (high persistence A), a protein that mediates multidrug tolerance through a mechanism known as persistence.  Essentially, during times of stress, such as the presence of antibiotics in an environment, HipA causes bacterial cells to enter a dormant state in which all cellular activity stops.  During this time, antibiotics are not effective against them because the functions targeted by antibiotics are shut down.  After a period of time, the levels of HipA in the cell decrease, and the cell returns to normal functioning.  These cells are known as persisters because they survive antibiotic treatment.  You can learn more about bacterial persistence here.  The Brennan lab is collaborating with a drug discovery company to find molecules that bind with HipA, potentially reducing its ability to induce dormancy.  In the future, these findings could lead to improvement of antibiotics.

My project involves isolating HipA from E. coli that are engineered to overexpress it, or make much more of it than they usually would.  HipA is a kinase, which means that it transfers phosphate from ATP to other molecules, and its activity can be measured by its ability to autophosphorylate, in which it uses its kinase abilities to phosphorylate itself.  I can treat HipA with the drug precursors to determine if any of them can inhibit HipA’s autophosphorylation, since if HipA cannot phosphorylate itself, this is an indication that it has lost its function as a kinase.  My longterm goal is to determine which, if any, of the drug precursors inhibit HipA’s function.  You can read more about HipA and phosphorylation here.

Field trip to the lemur center!


Science smells like blue cheese?

I can’t remember how many times Dr. G said, “Your first experiment is going to fail,” at our first two meetings.  These words went right over my head, and I definitely didn’t understand what he was talking about until Wednesday, when I was finally allowed to do something in lab by myself.  After two full days of following my postdoc around and being shown so many new techniques my head was spinning, I was told to repeat the first procedure I was shown, creation of a culture medium for E. coli.  Equal parts excited and terrified, I marched off to the cell growth room, dumped 37.5 g of agar in six flasks, added 1.5 liters of water, and autoclaved them.  I was proud that I successfully navigated the autoclave, a huge, intimidating machine that generates a lot of heat.  An hour later, when I removed my flasks from the autoclave, I noticed they were a funny color and producing bubbles.  Hmmmmm.  I asked my postdoc about it, but he thought everything was fine.  The next morning, I came back to a huge mess.  The flasks, which should have contained a light brown liquid, were cloudy and full of clumps of agar.  My postdoc quickly realized my error: I had used LB agar rather than LB broth.  Both bottles are stored in the same cabinet and filled with powder of the same consistency, color, and smell, and I had picked the wrong one.  Uh oh.  Now I had created a huge mess and couldn’t grow the cells from the preculture I had prepared.  All was not lost, however, because I borrowed some media from another lab member.  Long story short, the E. coli I placed in the borrowed media grew too slowly at first and then so quickly that they became overgrown and had to be bleached.  My first experiment had failed before I even got past the first and easiest step.

This early failure revealed a lot about what lab work is like.  First, I’m going to make many, many mistakes.  I’m incredibly fortunate to have a really kind and understanding postdoc.  His patience with my endless questions and general cluelessness about everything in the lab amazes me, and I could not appreciate it more.  As long as I learn from these mistakes, they’re not time wasted.  I can promise you that I will never, ever use LB agar instead of LB broth again.  Second, science doesn’t always work in real life like it does in the textbook.  Sometimes bacteria won’t grow for hours and then multiply too rapidly for reasons unknown to us.  Sometimes you’re working with a “fussy” protein that doesn’t behave the way it should.  Sometimes a protocol that has worked 50 times in a row fails.  There’s a lot more trial and error involved than I was expecting, but again, I’m lucky to be working with a very patient postdoc who has created well-tested procedures.

Two of my goals for the summer are to learn from my mistakes and embrace the uncertainties and questions that accompany research.  I also want to be patient with myself.  I have learned an insane amount of techniques and procedures in the past week, and I forget the details almost immediately after I learn them.  It will take me at least two or three tries (probably many more) before I feel really confident doing something, and that’s okay.  Finally, I want to get to the point where I can walk into the cell growth room without feeling knocked off my feet by the overwhelming stench of bacteria, something that smells to me like bad blue cheese.  All of these goals will be accomplished with increasing hours spent in the lab, and I am ready to have an exciting, surprising, educational, and stinky research experience!