Author Archives: Luke Sang

Much More to Discover and Learn!…

The past eight weeks in the BSURF program have truly been amazing. My time in the program and in the Brennan Lab has exceeded all my expectations coming into this summer. I remember that some of my most important goals coming into this summer was getting a better idea of what I wanted to study, do meaningful research, to build a bigger network, and lastly to bond with other students in the program. Although eight weeks is still a relatively short amount of time, I felt that I truly got to take great strides in these regards, if not successfully complete them.

Tackling the idea of figuring out what I wanted to study was something that was made more clear to me through my own research and also the guidance of my mentor and guest speakers. I’ve always felt that a lot of things in science were interesting and that if I set my mind on any one subject, I would eventually be passionate in it. During this summer, with the abundance of guest speakers, I sort of got to test that theory out. Listening to speakers investigating a wide range of sciences allowed me to get a better idea of what I wanted to study. I found that while certain topics of science were interesting, they didn’t quite align with the way my curiosity was urging me to investigate. Growing up, I had always been a visually learner. If I could see it happening and see the moving pieces of why it was happening, that helped me better understand a certain idea. This exact concept was practiced upon in the lab I worked on this summer. X-ray crystallography in the Brennan Lab was an interesting concept that while I had heard of, never had the chance to deeply explore until now. Working to build structures of protein and seeing how certain sites interacted with other molecules was really fascinating. And while my research this summer was attempting to progress towards possibly solving a structure, building from the very base of expression, this type of thinking gave me an idea of what I wanted to study in the near future: attempting to build structures and models of proteins and see how they interact with other molecules. This curiosity also steers my courses towards a more biochemistry focused schedule, somewhat clearing up my dilemma of what to study. I also still do believe that a scientist will go through many phases of wanting to learn about many different things, leaving the future with exciting possibilities.

I came into this summer unsure exactly what “meaningful research” meant. From an outside perspective, it seemed that all meaningful research had to be groundbreaking or on a “hot” topic. This summer has completely changed what I define as meaningful research. I’m sure this definition will change many times as my career evolves but currently meaningful research to me is simply research that allows me to deeply learn about a topic I find interesting. Hands-on work and visual work has always been a dimension of learning that I haven’t been able to experience outside the classroom, so through this summer my work has certainly been meaningful. Further, I will also add that working towards a larger goal, in this case solving a protein structure, certainly brings meaning in my own mind. Being able to possibly contribute knowledge to other and also learn to myself is definitely meaningful to me.

The variety of individuals in science I have met this summer has truly expanded my perspective and also my network. Even a simple conversation or listening to a new speaker allowed me to get an idea how different scientist think and approach their work. Throughout this program an emphasis has been put on communicating work and that ties directly into interacting with other scientists. The amount of researchers at Duke I have met this summer as definitely given me a better idea and perspective of the landscape of research as a whole here at Duke.

Finally, this summer has allowed me to build friendships with other students in the program. With everybody doing challenging research, getting lost in literature papers and being overwhelmed by lab protocols has definitely humorously brought us all closer together. This is possibly the most rewarding part of this summer as these friendships were certainly last as all of us continue our time at Duke.

I’d personally like to thank Dr. Brennan for allowing me to work in his lab this summer. It has been a wonderful experience and I am very much excited to continue working next semester. I’d also like to thank Grace for her mentorship and her patience to help me throughout my learning process. In addition, I’d like to thank all the members of the Brennan Lab for creating a friendly and learning environment and also for helping me whenever I asked a question. Dr. Grunwald and Jason were also crucial to my growth and experience this summer and I would like to thank them for their time and sacrifices.

And for the last time,

Thanks for reading!


Luke Sang

Investigating the Microbiome – Dr. Lawrence David

Over the course of the program there have been many great talks that have given me a glimpse into various topics of research. Speakers have also shared their own personal journey and wisdom so that it might help younger scientists, like myself, to possible get a better understanding of what they might want to do. The talk that stood out the most to me in both topic and experience was that last one given. Dr. Lawrence David recently gave a guest lecture to us about his work with nutrition and his own journey to become the scientist he is today.

One of the things that stood out most to me about this talk is Dr. David’s admittance that many times along his journey he wasn’t sure what the “right” path was or what he wanted to do. Instead, he emphasized a message sticking with something and keeping an open mind to new ideas. When he had to decide whether to go to graduate school and continue to pursue research, he chose the continue on that path because it “wouldn’t hurt.” The idea of undertaking a path because it leaves the most options open is actually something that really resonated with me. Most of the time, we don’t know if we have made the right decision until after situation has been played out. Personally, I am not completely sure what exactly I want to study or what exactly I want to specifically pursue in the future. To receive this type of advice and hear about this type of experience allows me to better be prepared as I move forward and explore my interests. Dr. David sharing his real experience and advice was really helpful to me as I am still figuring out what exactly I want to study in the future and pursue. The concepts of keeping an open mind and sticking with something even if you don’t like it at first are two important concepts he constantly talked about that will definitely be on my mind as I move further into my academic career.

Furthermore, his study on the human microbiome was also very interesting. His work seemed very applicable to real life as people are constantly trying to be aware of what they eat. Dr. David’s work on better understanding how to optimize certain diets and nutritional supplements leads to more research on how humans can better take care of themselves and improve their health. I found his work particularly interesting because of how applicable and just commonly “found” it is. People make these decisions on their diet everyday, and research like his allows people to better improve their everyday life.

Dr. Lawrence David’s guest lecture was extremely helpful in his practical advice he gave about approaching science. Because he was only a few year removed from his schooling, his advice was prevalent and resonated with me. The sharing of his own experience and research definitely encouraged me to continue on the path of research.

Process and Abstract…

So far during my summer of research there have a combinations of highs and lows. Leading up to now, much of my research has been spent in trying to troubleshoot the issues with my project and trying countless different approaches to attempt to successful express and purify my protein of interest. Needless to say, there were definitely some frustrating days where after a week of work and anticipation resulted in a negative result. I can proudly say I have found many ways to NOT express my protein of interest. Yet even in frustrating times there were many lessons to be learned and interesting topics to understand. Often times scientists talk about how they learn the most from their failures, and this concept has never been more evident to me.

However, not too long ago I experienced the opposite of such valleys. Having tried a new bacterial strain and additive of glucose to my cultures, I was able to express and purify my protein. It was definitely a great feeling to see positive results and to see the product of countless hours of work. This result now allows me to continue moving further into the project and approach the ultimate goal of solving a structure. With the protein expressed, I can now run different assays and experiments to further test the reactivity and nature of this particular protein.

Seeing both sides of the story in research, in failure and success, has definitely made me appreciate how much there is to learn in both regards. Failure brings knowledge and information that success can’t, and same vice versa. I look forward to continuing my project and experiencing the joys and woes of research to come.


***This is a draft, revised version emailed***

What is the structure of RAMOSA3 and what are its implications outside the trehalose pathway?

In corn, RAMOSA3 (RA3), a metabolic enzyme, is responsible for the dephosphorylation of trehalose-6-phosphate to create trehalose. Mutated versions of the RA3 protein have shown phenotypic differences in the development of corn, creating long branches at the base when compared to the wild-type corn stem. However, it is still unknown how altering this particular protein ultimately creates these phenotype differences and what exactly are the moonlighting capabilities of RA3. We suspect that RA3 has transitional regulation capabilities, whether through the regulation of RAMOSA1 (RA1), a known transcription regulator, or by its own means due to its acting upstream of RA1. We attempted many different approaches to express the plant protein RA3 into different strains of Escherichia coli (E. coli), testing many strains to find one most optimized for creating such protein. Further, we tested different conditions, altering temperature and time, in which to grow the bacteria in order to optimize protein expression. We also created a custom purification process best suited to isolate the protein of interest to run assays to test its function. Lastly, X-ray crystallography will be utilized with successfully grown crystals with RA3 in order to solve a structure. Our findings suggest that the C43 (DE3) strain of E. Coli was best suited to express this protein after being grown for three hours after induction at thirty-seven degrees Celsius. A purification process focused on the histidine tag within the protein was successful, as a process of a nickel column combined with imidazole washes isolated the protein. *Future assays and advancements in the project will allow us to reach further conclusions. At the moment, our results allow us to suspect that it will be possible to solve a structure of such protein and possibly create crystals to perform X-ray crystallography. Our future conclusions will allow us to confirm the function of RA3 as well as further develop the possibility to solving a structure.


*Assay results will be ready by poster presentation, along with possible other advancements into the project


Revised Abstract:


Investigating the possibility of solving a crystal structure RAMOSA3 through analysis of expression and enzymatic activity.

RAMOSA3 (RA3) is a metabolic enzyme in maize that is responsible for the dephosphorylation of trehalose-6-phosphate to create trehalose. Mutated versions of RA3 have resulted in phenotypic differences in inflorescence branching at the base of the axillary meristem within maize. However, the transcription regulation functions of RA3 and its signaling interactions with other molecules are still unexplained, making a solved crystal structure vital for further knowledge. This study investigates the possibility of crystallization through expressing large amounts of RA3 in bacteria, purifying the protein, and confirming the function of the isolated protein. We tested expression of RA3 in multiple Escherichia coli (E. coli) through Isopropyl β-D-1-thiogalactopyranoside induction and variating temperatures and growth time. Then, we designed a purification process targeting the hexahistidine tag in RA3 to isolate the protein. Sentence about assay here. Our results show that RA3 was most optimally expressed in the C43(DE3)pLysS strain of E. Coli at 37 ℃with a 3 hour growth time and isolated with a NiNTA Agarose column followed with step elution of imidazole. Sentence about assay results here. The study suggest that crystallization is possiblewith the condition of (conclusion depends on assay results).


Three Days in the Lab!

So far through this summer, I have discovered that the stereotypic perception of the routines of a researcher is mostly false. Each day there is usually a new challenge, concept, or method to try, creating an experience very much different than the monotonous routine everybody is seemingly attaches to research. It’s true that a simple task of pipetting or waiting for a culture to grow may seem repetitive, but all these things ultimately build up to contributing towards a larger goal which makes even a daily routine exciting. As I move further into my project, each day is new as there is a new step to be taken in ultimately solving a structure for my protein of interest. The things done in the lab are often determined by the results received the day before, as I have had a wide range of different days in the lab. However, here is a slight look into a possible day in the lab.

If I was just starting a new experiment or starting from the very beginning to express a protein and eventually solve a structure, I usually start off with checking my bacteria cultures from the night before. After checking the cell density of these cultures, most times I dilute them and wait till they grow toward the preferred density for the experiments of the day. While that is happening, depending on the day, I will either prepare needed solutions or samples needed for the rest of the day. After the bacteria has grown to preferred density, I usually will conduct an inducing experiment and attempt to express the protein of interest in different conditions. Often times there are time conditions involved in these different conditions so some of the day does consist of waiting. After waiting (this could be actually be done the next morning, depending of the conditions), I run an electrophoresis gel to see the results of the conditions. Analyzing these results will give me indication for what my next few days will look like. If there is significant protein expression, the next few days will consist of creating large cultures of the same strain of bacteria with the protein of interest in order to attempt a purification process. The purification process is generally consists of lysing the cells and running a column to ultimately elute the cells. If not, then it is back to the drawing board, trying new strains or methods in order to express the protein needed. I would create new cultures and grow them overnight to once again begin this process all over again.

Often times it seems like my “day” is actually three days in total as these three days is what it takes to usually complete a cycle of experiments (growth, expression, gel/purification). With results, my following day would be quite different than the preparation days. In addition, because I am now helping with a project analyzing the protein MprA, my days now also consist of ITC and other purification methods. The exact details of a day are often driven by the results of my previous experiments, leaving every day up in the air and adding an element of excitement.


Till next time,

Luke Sang

A Different Approach…

My week was highlighted by great chalk talks from every member of the BSURF program. I definitely learned a lot about various topics and projects. Further, these chalk talks spark my interest in new subjects and has gotten me talking about things I normally would have never even thought about! This week was a great lesson in the communication aspect of science and research: having to prepare a chalk talk, listen to presentations from others, and explore other topics through challenging question has certainly helped me to see the utmost importance communication and discussion has in research. Something to be noted is a question asked during my own chalk talk as given me a new idea in which to approach my project, something that might actually help move along my project objective.

That being said, a talk that particularly stood out was the one presentation by Simeon. Simeon gave a chalk talk on the study of essential genes involvement in cryptococcus neoform sexual reproduction expression.

He began with a bit of background information, presenting the case that cryptococcus is a fungi that can cause brain damage to those suffering immunodeficiencies. Cryptococcus is specifically hard to target and create successful drugs to combat it as through its sexual reproduction process different and various types of the cryptococcus fungi are formed and some of these will ultimately carry drug-tolerance. Therefore, going into his project, Simeon noted that he is targeting two essential genes involved in sexual reproduction. Using different constructs, the goal is to test three different approaches and see which conditions will ultimately repress sexual reproduction the most. Having more information on what successfully alters the reproduction expression within these fungi could build toward antifungal treatments in the future.

I found this presentation to be particularly interesting due to the way in which they approached this drug-tolerance issue. Approaching this issue from the perspective of altering sexual reproduction creates an opportunity to then create drugs to target specific types of cryptococcus to combat this fungus. Other studies focus on functional proteins within the fungi or creating drugs that will be undeterred by such tolerance, leaving this approach quite interesting. As this fungi issue is a big clinical problem around the world, this different approach could ultimately begin to lead toward a solution to this problem.

I am very much looking forward to see the information or conclusions gathered by this project and others at the end of the program!



Luke Sang

Get To Know Dr. Brennan!

Dr. Richard Brennan began his undergraduate studies at Boston University where he was initially a chemistry major, but eventually transitioned to studying biology. Dr. Brennan fell in love with studying English and communication, leading him to a minor in history. Till this day, he emphasizes the importance of language and communication within science and advocates for all scientists to become trained writers. He noted to me that during his undergraduate years he found that science is just as creative as the arts, furthermore, scientists are creative in a unique way that blends subjects from multiple areas. This passion led him into research the summer before his junior year where he was part of a fellowship research program. The creativity in the lab instantly drew him towards research, and during his junior and senior year he worked in a biochemistry lab. In some sense, Boston had become the perfect place for him to begin his research career as it offered rich history and advanced resources in research.

After his undergraduate studies, Dr. Brennan attended Cornell to start a Ph.D in biochemistry. However, as he went straight into graduate school without a break, he decided to take a year off and research at biochemical genetics lab with the purpose of going back to school in a year. Through this process he came to become very interested in structural biology: throughout his earlier years he had worked on a project involving proteins that centered around the develop of a male birth control pill, and he came to find he interested in being able to see the things he was studying. With that interest, he found a home in the University of Wisconsin which was, and still is, one of the leaders in x-ray crystallography research and biochemistry. Here, Dr. Brennan did a lot of work with creating structures for long DNA and made restriction enzymes. After his work in Wisconsin, he chose to set out to the University of Oregon where he solved a structure of a protein DNA complex (only 3 or 4 other known structure at the time!). He was faced with a decision to take a job in Boston or in Portland. Eventually he chose to work at the Portland Medical School where he would spend 20 years, become an endowed chair, and build a strong program. After some time, he moved to MD Anderson in Houston where he was tasked with strengthen their biochemistry department. Here, his program did extremely well in their research and scientific endeavors; however, due to academic structure, it was difficult to attract students. Then one day, Duke University contacted Dr. Brennan about becoming Chair of Biochemistry. Dr. Brennan has since been the Chair of Biochemistry at Duke as of January 2011, where he continues to do research.

I asked Dr. Brennan what his goals were along the way and whether he experienced any hardship along that way that might have altered them. The over-encompassing question he was interested in figuring out was how things work. His focus in structural biology allows to develop and understanding in how certain functions work and develop a deep understanding in the “why.” Further, Dr. Brennan had a goal on figuring how to see why certain things worked the way they did. This fit the idea of structural biology and how solving a structural allows you to truly understand why it works the way it does. Without the basic information on what something looks like, you cannot do any further experiments or research with this specific thing of interest. His goal was to apply creativity in research in order to answer these questions.

Yet, Dr. Brennan accounted to me that during his Postdoc work, he became curious with the idea of leaving academic science and turning toward a career in scientific writing. And so, he interviewed for the head editor at a science journal. He was selected for job and was forced to make a decision. He evaluated which road would give him the most creative freedom as that was of utmost importance in his research and academic endeavors. Eventually he chose to stick with academic science as it offered him the best route to continue answering his questions, and he could also continue to write within academic science. To this day, Dr. Brennan has continued his work in research and contributing towards the structural biology field. Dr. Brennan is currently interested in studying how multi-tolerant cells work and how activation signals are triggered.

Dr. Brennan has continued to share his perspective in the creativity of science through teaching at Duke University. He enjoys teaching due to the interaction and opportunities there are to inspire future scientists. He believes that individuals should teach at schools with high-end resources as it’s important to share what you’ve learned others.

As one final piece of advice, Dr. Brennan warned against the elitism in science. Very much discouraging this attitude, he cites the stereotyping when it comes to choosing universities and places to study. Often times scientists miss out on opportunities because they make choices with predetermined ideas. It’s important for scientists to adopt an open-mindset and one that truly values everything on the same playing field.


Luke Sang


Plants Meet Fungi!

As I mentioned before, I am currently working in the Brennan Lab, a Biochemistry lab that has previously done extensive research into the trehalose pathway within fungi. In order to lead into my project, it is important to understand more about certain research in the Brennan lab and what exact implications it has in the large scheme of things.

Throughout recent times, immunocompromised host populations, whether transplant recipients, cancer victims, and other ICU patients (Intensive Care Unit), have continuously been invaded and killed by fungal diseases. In a search to identify antifungal targets, the Brennan lab investigated the trehalose pathway due to its fungicidal characteristics, broad relevance to many different types of fungi, and its low toxic consequences in mammalian species.

Looking a bit closer into this research, the trehalose pathway in fungi is regulated by two important enzymes that basically form the backbone of trehalose formation: Tps1 (trehalose-6-phosphate synthase) and Tps2 (trehalose-6-phosphate phosphatase). Tps1 creates trehalose-6-phosphate (t6p) from glucose-6-phosphate while Tps2 ultimately forms trehalose from t6p. Trehalose is extremely important in fungi survival as it has implications to energy reserves during times of stress and is crucial to carbon metabolism and regulation. Tps2 has been identified, due to structural research and animal tests, to be an ideal fungicidal target as mutant Tps2 fungi cannot survive in high stress environments. However, recent research has also uncovered that Tps1 and Tps2 seem to serve other functions within fungi than simply the trehalose path way, signaling that these are possible “moonlighting” proteins.

Well, you’re probably wondering, how does all of this relate to plants and my project? Recently, collaborators of the Brennan lab have studied a protein in maize, RAMOSA3 (RA3), that is part of the trehalose pathway in plants. RA3 serves the purpose of converting trehalose-6-phosphate into trehalose, an analogous function to that of Tps2 in fungi. Yet, when RA3 was mutated in maize, the phenotype of the maize changed and at the stem of the maize, and weird abnormally long branches would begin to form. This gives some indication that RA3 is much more than just a regulator within the trehalose pathway. Once again, this RA3 enzyme is suspected to have possible transcription regulation implications. My project is focused on taking the plasmid containing the RA3 protein and materials sent by collaborators to express the protein, purify it, confirm its function, and ultimately create a structure of it. In order to better understand the functions of RA3 and its implication or analogous functions to the possible fungi trehalose path, we need a literal structure of the enzyme to study substrates and other similar patterns. The moonlighting similarity is yet another thing to be studied. As RA3 is relatively still not understood, my project is focused on create a better familiarity with this enzyme.


Till next time,

Luke Sang

Lots of Questions…

This summer, through the 2018 BSURF Program, I will be researching in Dr. Brennan’s biochemistry lab. Specifically, I will also be working with a graduate student in his lab, Grace. Previous to this program, I had only really done wet-lab work in courses, college and high school, where students simply followed instructions and completed a lab report. This experience would be my first time independently researching within a lab, creating a sense of excitement, uncertainty, and curiosity within me.

Primarily, I am expecting to learn a lot about lab procedures and the topics relevant to my specific focus in biochemistry. As mentioned before, as this is my first official lab experience, I am excited to get a glimpse into the life of a researchers and how he or she goes about answering a scientific question. The basic lab procedures, writing in a lab notebook or how to develop certain solutions and results, will be knowledge for me that will be essential in the future. I am curious to know the “why” behind everything, being prepared to ask questions that helps me develop a better scientific knowledge and basis for why certain things are done. I am hoping this knowledge helps me gain a better understanding of the things I have learned in the classroom, as well as preparing me with concrete methods and approaches to solve problems in potential future research. Whether it is seeing why researchers in this lab use certain antibiotics of a specific chemical make-up compared to another or why an environment induced better protein expression than another will allow me to apply all those “textbook” concepts I learned in past courses and develop a new perspective on them.

Expanding off just gaining more knowledge, I am looking forward to this summer as a place where I can start solidifying what I will be studying or majoring in the future. Personally, I learn better in environments that are hands-on and places where I can make mistakes and discover more about the topics I am applying. This experience will allow me to see if Biochemistry or chemistry is a possible route of study I would enjoy, as I ultimately see myself applying the concepts in a classroom rather than just simply learning them. Working in this lab will give me an idea of what a specific learning path will lead to.

Lastly, I am definitely excited to meet new peers, mentors, and friends along this summer program. Seeing as researchers collaborate a lot, building a relationship with those in my lab and within the program is something I will be focusing on. Getting to know both Dr. Brennan, the graduate student mentoring me, and other people in the lab is something I can’t wait for. Furthermore, students in BSURF are a collection of some of the most intriguing and friendly people to get to know better. Other than that, people here still at Duke, such as the Huang Fellows, are more students to get to know better. Building these new relationships and networks will certainly make this summer quite the experience.

Till next time,

Luke Sang

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