Category Archives: BSURF 2018

Throughout my eight weeks as a BSURFer, we had various faculty members present their research along with their inspiring stories about their journey to a faculty postition. An overall theme in each of their stories was the uncertainty about their path in life. Most of the professors had never thought about entering academia and a few had gone to medical school only to fall in love with research later. Of these faculty seminars, my favorite was Nutrition: the Human Microbiome (and me) by Dr. Lawrence David.

Dr. David’s research focuses on how dietary compounds stimulate growth and metabolism of gut microbes. Previous experiments explored the differences between high-fiber plant-based diets and low-carb animal-based diets through the analyzing of stool samples. Currently, his lab is investigating the most beneficial pre-biotic supplements to feed the microbes in a human gut. Since a large part of staying healthy and preventing disease in a majority of the population is based on diet, the experiments conducted in the David Lab are important for the overall health of the masses.

While his research was amazing to learn about, the advice portion of his presentation is what impacted me the most. He advised us to stay with our undergraduate lab for a long period of time, because good science can’t be done in just a semester. He also discussed his experience with graduate school because though he had some uncertainties at first, he realized that the experiments he was conducting made him happy and he enjoyed the process of scientific research. His final piece of advice was about knowing when to graduate. Once he finally felt like he knew what he was doing and became comfortable, his PI told him it was time to graduate. When he felt comfortable in his understanding of research, he stopped learning new things and stopped growing as a person, so it was time to take on a new adventure and learn something different. This constant sacrifice of intellectual comfort for new experiences is the difference between good and great, so I’ll be sure to carry that piece of advice with me through the rest of my journey in science.

Throughout the course of the summer we have listened to talks from professionals from all sorts of backgrounds and focuses particularly in the field of science. We were able to here from multiple perspectives as to how these medical doctors, researchers, and some medical doctors/researchers achieved their goals in science and even what they are doing to reach their goal. It was interesting seeing how two people could end up at the same point but take two totally different paths to get to that point and it really helped me to understand that my path is not set in stone it is free to change as I discover my interest and what I want to do in the future.

With all that being said I have to say the my favorite talk was most definitely Lawrence David. He seemed to struggle  with the same problems that I am tackling as an undergraduate currently, which made his talk very relatable. I found his research about “Nutrition and the Human Microbiome” interesting and it may be something that I want to look into in the near future. Through his stories about how his interest change from possibly wanting to go to medical school thank deciding to do research, I realized that my interest may change and I should be open to that change.

I found his advice on how to choose and undergraduate lab very helpful. I was struggling for the longest time trying to decide if I would stay in the lab I am in now, move to a different lab, or try an new experience all together. I realized that no matter what I would be gaining experience and that is what is most important.

I definitely see him as a possible mentor to help me make some important decisions in the near future!

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.

One of my favorite activities of the BSURF program was the opportunity for all of   my classmates and I, along with Dr. Grunwald and Jason, to gather in the early hours of the day to listen to inspiring scientists discuss their research focus and their academic path towards this focus.

From Dr. Schmid’s focus on extremophiles, to Dr. David’s focus on microbiomes,  I learned so much about aspects of science that I had never even heard of before. One faculty talk that really intrigued me was Dr. Noor’s talk on evolution and genetics. In his talk, Dr. Noor explained that evolution is comprised of two processes: changes within a current lineage and formation of a new lineage. He then discussed one of his current research ideas about recombination and what happens to differentiation of species if recombination is stopped. He studies this in Drosophilia (fruit flies). From my understanding, one of his broader goals is to examine how new species are formed and how they are sustained through genetic inheritance.

One thing I really liked about Dr. Noor’s faculty talk is his ability to explain his research project in a fun, interesting, and lively way. He also really brought to light the intersections of science and society through his discussion of the current political climate on evolution. I could definitely understand his concern about the lack teaching of evolution in grade school, having seen this phenomenon in my own high school biology class. Another aspect of Dr. Noor’s talk that I really enjoyed was his discussion of evolution as it applies to other sciences, including medicine. Specifically, he discussed how evolution could explain antibiotic resistance to bacterial diseases/ infections, as well as mosquito-borne diseases. Prior to this, I had never even know that this was a growing problem. Overall, I think it was really interesting to see how Dr. Noor’s research can help increase our understanding of evolution. Hopefully I can have a chance to take one of his classes in the future!

Having all these seminars this summer has really made me appreciate the huge variety of faculty at Duke and how amazing many of the people here are. The fact that I can contact many of them and have a discussion is even more extraordinary. As a result, I find it difficult to select just one presentation that I found the most fascinating, and instead I wanted to focus on my two favorites and how the ideas they presented have overlapped in my mind.

First was Dr. Mohamed Noor’s research on the sister species of Drosophila, D. simulans and D. pseudoobscura, and how they have very similar songs that are slightly modified to avoid interbreeding between the species. This got me rather curious about fruit fly songs and so I took a cursory look around at what had been published. I was really surprised to learn that fruit flies aren’t born knowing what song to sing, but instead they learn it from other flies around them (link). Yes, you know those small things that, no matter how many times you swat them away, will return time and again to annoy you while trying to enjoy your lunch? Apparently, they actually can learn and they’re just terribly rude. But in all seriousness, flies can learn! Something that appears so simple and lacking anything beyond what its genetics programmed for it is actually notably more sophisticated. Perhaps I was just underestimating other animals, but when one is often thinking of organisms at the cellular and genetic level and functioning more as biological machines, sometimes one forgets about the complexities and capabilities of the whole creature.

Secondly, there was Dr. Steve Nowicki’s research on bird song and what it reveals about animal communication and behavior. In particular, I was fascinated by this idea that behavioral responses were caused by stimuli that are sorted into categories as opposed to a linear relationship between the stimulus and the behavior. Like the above, it’s something that I hadn’t ever considered since it always just seemed logical for behavioral responses to be linear. However, reflecting on this new insight, it actually makes sense for it to be categorical since the brain often cut corners where it can and categorization would be an easier system for it to implement compared to having a wide array of responses to a spectrum of intensity of a certain stimulus.

Now, how do those to come together? Well, I’ve been thinking a lot about how categorization may occur in intraspecies interactions besides in birds and humans. Then I thought back to Dr. Noor’s seminar and began wondering if there was categorization occurring in the sister fly species and if changing the duration, frequency, and order of the “notes” in the flys’ songs could change the females likelihood of accepting or rejecting the male, and if the change would look like the categorization that Dr. Nowicki was seeing or if would be more linear. Whether or not that’s an idea worth looking at, I don’t know, but I find it kind of interesting.

Overall, I really enjoyed all the seminars over this summer, though I must admit that I found myself most interested when our speakers were discussing the work they had done or are currently working on. However, the seminars have gotten me to really consider the idea of pursuing a PhD, and there are a few words of wisdom that were shared that I expect to be keeping in mind as I go through my future career.

Throughout the past 7 weeks, the research fellows have had the great privilege of listening to some amazing faculty speakers. From Dr. Nowicki investigating bird song to Dr. David collecting fecal matter for microbiome analysis, I have thoroughly enjoyed listening to the exciting research Duke faculty are doing and how they ended up doing research. One specific talk titled “Epigenome editing in neurons with dCas9 fusion protein” by Dr. Anne West really caught my interest because of both the science and the life lessons shared during the one hour she had with us.

Dr. West spoke about the changes in gene expression when neurons are fired and how the environment can play a role in DNA methylation which results in a changes in the epigenome. She talked about using the new technology of CRISPR-Cas9 to aid her research and emphasized that research is heavily based on the tools available at the time.

Although I found her science very interesting, her final note was what really stuck with me. She shared with us her lab’s mission statement which is:
“To answer vigorously defined questions,
To reveal something new about the brain,
To enjoy the process of discovery.”

These statements allowed me to reflect on my own choices when doing research. The first statement about answering questions emphasizes both the importance of asking good questions and having the resilience to answer them. The second statement highlights the importance of doing novel research rather than repeat experiments. The third statements underscores the fact that research needs to be fun and it is pointless to do something that isn’t. Research consumes a lot of time, energy, and resources so it is important to enjoy the process. These statements have made me realize that research is probably something I do wish to pursue because I enjoy spending time in the lab and asking and finding answers to relevant questions.

Dr. West was an amazing and inspirational speaker and I hope that I can take the lessons she shared and use them to motivate me in my path to pursuing research in the future.

In my experience, it’s not often you meet someone who works with poop regularly. So when I learned that Dr. Lawrence David studied the microbiome using human stool samples, I was immediately intrigued. But it wasn’t Dr. David’s research that made me choose to write this blog about him. Instead, I appreciated how honest and open he was about his career path.

I felt like Dr. David’s background on his undergraduate career was very relatable in some aspects. During his undergraduate studies as a Biomedical Engineering major at Columbia University, Dr. David was immersed in an environment where majority of his classmates had medical school on their minds. As a prospective biology major with no intention on going to medical school, taking courses alongside pre-med students is something I’m familiar with. With medical school off the table, I plan on going to graduate school. And unlike those who went to medical school, Dr. David decided to attend graduate school, which I think proved to be a great decision for him.

While working towards his PhD in Computational and Systems Biology at MIT, Dr. David studied the microbiome by tracking the bacteria active in the gut. From how he presented his experiences, it seemed like he finessed his mentor to fund his trip to Asia alongside his partner. Since he was eating meals different from meals in the US, this change was reflected in the bacterial activity in his gut, which he was able to track after collecting fecal samples throughout his trip. (And fortunately, he saved an image of his fridge full of samples as proof!)

Despite being a relatively young scientist, Dr. David’s talk was full of insight and helpful advice. He found that making short-term decisions worked out well for him, and what ultimately matters is whether you’re happy in the present moment, not whether you’ll be happy 10 years from now.  A part of being happy is getting to know others while doing something fun. While it’s great to be passionate about your work and dedicate a lot of time to it, it’s just as important to have other hobbies and socialize with others in a non-professional environment.

One of the biggest takeaways from this talk was to do something unique. During your career, you may have some moments that could be weird. But based off of Dr. David’s experience, these weird moments can be monumental and influence how you continue progressing in your career. And even if things don’t turn out like you expect, at least you have unique memories along the way.

In the past couple of weeks, we have had a lot of amazing, accomplished speakers, all at different points in their careers, come talk to us about life and science. Each talk has been fascinating, with a new life story and lens to look at research every time.

Of all the talks, the one that stuck with me most was given by Dr.Mary Klotman, as she gave some really interesting insights into HIV research. Previously, when learning about HIV, I always looked at it from a social perspective, rather than a scientific one. Thus, her talk taught me a lot about the mechanism of the disease. For example, I did not know that the virus uses kidney cells for long term storage. Additionally, I did not realize that the virus actually integrates into a hosts genome, making it difficult to find a cure. It was really fascinating to hear information about the symptoms and pathway of the disease from someone who has seen it in both a clinical setting and laboratory.

I think her experience with seeing patients living with HIV really made her talk stick with me. Her anecdotes about the experiences of patients, from those being taken care of by their mothers to those who killed themselves, made the talk even more impactful. She did research on HIV back when it was a guaranteed death sentence, and hearing how that impacted patients along with the science behind it made the talk a good blend of social and scientific.

Overall, Dr.Klotman’s talk on HIV was fascinating and informative. It was really a privilege to hear from someone with such breadth of experience as both a doctor and a scientist.

Abnormal and excessive tau phosphorylation commonly characterizes Alzheimer’s disease (AD) neuropathology through neurofibrillary tangles (NFTs) causing axonal dysfunction and microtubular instability. Essential to DNA repair, protein modification, and gene expression, the methionine cycle generates a methyl group upon the conversion of S-adenosyl-methionine to S-adenosyl-homocysteine, which can activate protein phosphatase 2A (PP2A) and leucine carboxyl methyltransferase 1 interactions in neurons.1,2 These interactions are known to target serine/threonine phosphate groups prevalent in hyperphosphorylated tau, and prior studies have demonstrated increased aggregate formation in vivo upon PP2A inhibition.1,2 Furthermore, increased levels of homocysteine, a byproduct of the methionine cycle normally converted back into methionine or anti-oxidative glutathione, is strongly linked to the progression of AD.3 This suggests insufficient methionine resynthesis allowing the hyperphosphorylation of tau to form NFTs. Biochemical analysis of the different pathways homocysteine metabolism undertakes show many similarities to digestive reactions in the liver of the reactive intermediate imine deaminase A homolog (UK114) enzyme. We are studying the potential interactions and role of UK114 in transfected Chinese Hamster Ovary cells by recording protein expression levels via Western blot. The transfected cells are subjected to various conditions either translatable with symptoms of AD or saturated with components of the methionine cycle that are potentially catalyzed by UK114. The results of these studies will further expand our understanding of the methionine cycle in relation to AD pathology, as well as explore the antioxidant uses of UK114 in an immunological scope.

Weekly Highlights:
*LOUD THUD as Dang accidentally knocks over a giant bottle*
“Is Dang throwing things again? Bad boy.”-Joan

“Let’s go stuff some squirrels and hamsters in a fridge to induce torpor!”-Dr. Colton
“We already tried that. It didn’t work.”-Joan
“PARTY POOPER!”-Dr. Colton

“Wow, big pellets! This sample is good!
*BCA subsequently fails. negative protein concentrations returned. half the trial ruined*
“Oh no.”-Huifang

“What’s wrong with your milk? :/”-Huifang as she throws away milk and goes to her own stash

“And one day, you’ll be like these two amazing children. One graduating with distinction and the other just a lowly freshman!”-Dr. Colton

References

1.Qian, W., Shi, J., Yin, X., Iqbal, K., Grundke-Iqbal, I., Gong, C., Liu, F. (2010). PP2A Regulates Tau Phosphorylation Directly and also Indirectly via Activating GSK-3β. Journal of Alzheimer’s Disease, 19(4), 1221-1229.https://content.iospress.com/articles/journal-of-alzheimers-disease/jad01317

2.Stanevich, V., Jiang, L., Satyshur, K. A., Li, Y., Jeffrey, P. D., Li, Z., … Xing, Y. (2011). The structural basis for tight control of PP2A methylation and function by LCMT-1. Molecular Cell, 41(3), 331–342. http://doi.org/10.1016/j.molcel.2010.12.030

3.Seshadri, S., Beiser, A., Selhub, J., Jacques, P. F., Rosenberg, I. H., D’Agostino, R. B., Wilson, P. W.F., Wolf, P. A. (2002). Plasma Homocysteine as a Risk Factor for Dementia and Alzheimer’s Disease. The New England Journal of Medicine, 346, 476-483.
http://doi.org/10.1056/NEJMoa011613

A majority of Duchenne’s muscular dystrophy cases are caused by a frameshift mutation in the DMD gene and the deletion of exon 51 with CRISPR-Cas9 can restore the reading frame in many patients. However, there are thousands of gRNA sequences to choose from with a deletion frequency of only 2%, so which gRNAs in introns 50 and 51 are the most efficient? To test this, I designed 10 gRNAs in both introns and transfected 293T cells with these gRNAs and a CjCas9 plasmid. I harvested these cells, ran a PCR with designed surveyor primers and ran these fragments on a gel. No new bands were seen that were different from my no-gRNA control lane so the gRNAs I designed did not make an indel. I will test 2 new gRNA sequences with the same method and expect to see two bands unique to the gRNA-transfected cells. The results from my experiment will be positive controls for my mentor’s project of developing a high-throughput screening method of hDMD-targeted gRNAs. With broader and quicker gRNA screening, the CRISPR-Cas9 tool will be more efficient and used as a human medical treatment option for patients with DMD.

Sea urchin embryos show a great affinity to replace cells necessary for normal development through a process called transfating in which cells in the embryo express genes of other cells to compensate for the removal. Specifically, the removal of primary mesenchyme cells (PMCs) which are crucial to the development of the skeleton triggers non-skeletogenic mesoderm (NSM) cells to reprogram in order to express genes found in the PMC gene regulatory network (GRN). A possible explanation is that the PMCs provide a signal to the surrounding NSMs that inhibit NSM transfating. This study seeks to determine 1) which genes are expressed in the PMCs and 2) what signal the PMCs provide to inhibit NSM transfating. Through in situ hybridization experiments using newly synthesized probes from a candidate gene list, we hope to determine which genes are PMC specific. Using in situ hybridization and drug inhibitor treatments, we test for ectopic cell reprogramming in order to determine the specific signaling molecules from the PMCs to inhibit NSM transfating. This study can broaden our understanding of the signaling molecules crucial to normal sea urchin development.

Cannabis is one of the most popular drugs consumed in the United States. While work has been done to quantify whole genome transgenerational effects of cannabis exposure, there has been little done to quantify transgenerational effects on a gene-by-gene basis. Previously, Reduced Representation Bisulfite Sequencing identified Shank1 and Dlg4, genes associated with the post-synaptic density and the post synaptic membrane, as being differentially methylated in the sperm profile of cannabis(THC) exposed rats and controls. It is however unknown that this difference in methylation is significant  under specific gene methylation analysis. It is also unknown if any differences in methylation cause significant changes in the expression of Shank1 and Dlg4. That is why in this study, potential differential methylation of Shank1 and Dlg4* were measured through pyrosequencing of the somatic tissues of F1 rats. In addition, RT-PCR** was conducted in order to quantify changes in gene expression of somatic tissues in these F1 rats. It was found that in Shank1 there was hypermethylation in both CpG sites of interests, while in Dlg4 there was hypomethylation in one CpG site and hypermethylation in the other.*** The next steps for this study would be to quantify the gene expression and methylation changes for the F2 generation, while also quantifying the phenotype in the F1 generation.

*Biotin labeled primer for this gene(necessary for pyrosequencing) may not arrive due to inconsistencies with Dlg4’s primers in PCR

** RT-PCR may not be done at the time of this poster presentation

*** This data is from the RRBS whole genome methylation, not from specific gene methylation from pyrosequencing

How does the ability to do phagocytosis of a new cell population compare to other dendritic cells?

The intestines are home to immune system cells whose role is to monitor the microbiota within the gut; one group of these cells being dendritic cells. Recently, a population of dendritic cells that are CD14+, a marker that was associated with macrophages, were found in the colon. The role of these CD14+ DCs in the colon and their ability to do phagocytosis is unknown. We hypothesize that that these novel CD14+ DCs will be better at phagocytosis compared to other DCs. We extracted cells from the colon and incubated them with a pseudopathogen with a colored marker under multiple different conditions. Afterwards the cells were stained with antibodies and analyzed using a flow cytometer. Under multiple conditions, we found a higher median fluorescence for the novel DCs when compared to the other DCs in the colon. At the same time, the novel DCs did not have a median fluorescence intensity as high as the macrophages. Our data suggests that the novels DCs have an increased ability to perform phagocytosis compared to other DCs but not at the level of the macrophages.

Dystonia is a neuromuscular disorder in which muscles make involuntary contractions, leading to abnormal and often times repetitive movements. It has been found that a common mechanism for several types of dystonia—each characterized by the gene adversely affected—was an impairment in ISR signaling. (Rittiner, et al,) Furthermore, the alleles causing dystonia have incomplete penetrance. Thus, individuals who have the dGAG mutation for dystonia could either be manifesting—individuals who exhibit the disorder—or non-manifesting—individuals who do not have the disorder but have the mutation. This project will focus on this idea of manifesting and non-manifesting patients with the dGAG mutation through the examination of exosomes and other extracellular vesicles. Specifically, this research explores the potential for exosomes to be biomarkers for the difference in manifesting and non-manifesting disease states, because exosomes are known for their role in intracellular communication and disease progression. To test this possibility, ultracentrifugation is being utilized to isolate exosomes from patient fibroblasts grown in conditioned media. Then, a bicinchoninic acid assay was run to examine protein content in both cell lysates and extracellular vesicle samples. For further testing, the plan is to run a western blot utilizing antibodies that will indicate whether or not exosomes were enriched, and then to send these samples to another lab for further testing. Potential results would be that there may be a difference in the RNA and protein composition of the exosomes between the manifesting and non-manifesting. There may also be a difference in the production of exosomes between the manifesting and non-manifesting. Finally, there could be no significant difference between these states.

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.

ABSTRACT:

***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).