The Unknown in Sorcerer’s Cave

Editor’s Note: Many of our students in ACU Biology Research work in teams composed of members with differing levels of experience. This blog was composed through a collaboration by Samantha Studvick (2nd year) and Kathy Le (1st semester) working together on the same research project.

Sam:  I began doing research with Dr. (Josh) Brokaw at the end of the Fall 2015 semester. Initially, I was on one of the Thomasomys phylogeny projects, specifically the CO1 (Cytochrome c oxidase I) group collecting mitochondrial DNA sequences. I worked on this research for all of the Spring 2016 semester, and I got to present at the ACU Research Festival and the Texas Academy of Science and Society for the Study of Evolution conferences.

Since I enrolled in the Biology Research course this semester, I began a new project expanding on the DNA sequencing skills that I had already learned. I was a little nervous, not only to be working on an unfamiliar project, but to be working on a completely new project in a newly formed lab team. Drs. Brokaw and (Diana) Flanagan created a new research group, under the direction of Dr. (Jennifer) Huddleston, for a project to identify a set of unknown cave bacteria based on phylogenetic reconstructions.

Example phylogeny of domain Bacteria from BMC Evolutionary Biology 2005, 5:34

Example phylogeny of Domain Bacteria from BMC Evolutionary Biology 2005, 5:34

A phylogenetic tree demonstrates the interspecific relationships between organisms (like a genealogy of species). Most phylogenies are based on DNA sequences, and this requires a complex process of DNA extraction, PCR (DNA replication), gel electrophoresis, DNA purification, DNA sequencing and alignment, and computer analyses. While the goal of the project was to begin identification, most of the semester was devoted to finding the correct method for DNA extraction and running the PCR.

Kathy:  In the fall of 2016, my sophomore year, I began my first semester of research with Dr. Brokaw. We did PCRs and ran gel electrophoresis data on bacterial cave swabbings done in Terrell County’s Sorcerer’s Cave (notably the deepest cave in Texas) with the goal of identifying the bacteria in this unique isolated environment.

Overall, we used three separate methods of extracting and replicating DNA because the DNA collected was not always able to produce bands. Even though much of this semester was devoted to finding a successful technique, I learned a lot about the processes behind running PCRs and gels. It’s quite interesting seeing something you learn about in a Gen Bio lecture class actually happen in the flesh. It makes concepts easier to understand because there is tangible evidence and processes that occur to support the concepts. For instance, the process of synthesizing and denaturing DNA. Eventually, we have enough to run a gel, since the DNA is copied to more than a billion times. Once this has occurred, we run the gel and search the sequence in Genbank to begin to identify the species of the bacterium.

Sam:  We first tried to transfer portions of bacteria colonies directly into the PCR tubes without DNA extraction followed by the basic PCR protocol. The results were unsuccessful, so we implemented a new strategy for DNA extraction. Again, the results were not really worthwhile. After altering some of the quantities for the master mix (combined chemicals used in PCR), we were able to get some results, albeit inconsistent. With the DNA isolated and amplified by our successful PCRs, we were able to identify three of the isolates as Pseudomonas, which gives us a starting point for next semester; however, we will likely be switching our DNA extraction/PCR protocol yet again to hopefully yield more successful and consistent findings.

Kathy:  Initially, it was a tough semester since I was coming in totally uninformed. I constantly had to learn as we went, continuing to ask questions about the protocol and materials used for PCR prep and gel-running. Gradually, I got the hang of it, as I’m sure everyone else who was a seasoned researcher in the lab had, since the research is basically student-run with supervision from their research advisors. My main goal was to just learn how to do the process and then become good at it. Seeing as we were failing to have bands on our gels, it was a bit discouraging to say the least. I felt that I was doing the procedure wrong or perhaps botching it on the filling of the wells in the agarose. We plan to correct this by using DNA extraction kits next semester to increase the likelihood of bacteria identification.

Sam:  Doing research can be a frustrating process at times, especially with ineffective protocol. Nonetheless, I really do enjoy conducting research with a team. As a hopeful Pre-Med student, this research is not directly related to my intended field; however, there are some implications as this project may begin to look into the development of antibiotic resistance (in a distant future). The techniques used in the lab are invaluable resources, though, and are important for a foundation in any topic of research. Being concurrently enrolled in Microbiology (lecture and lab) has broadened my appreciation of the study of bacteria. Moving forward, I am excited to continue working on this project, and I look forward to presenting my findings at various conferences.

Preparing bacteria for PCR for the last time in the Clark Stevens Lab.

Preparing bacteria for PCR for the last time in the Clark Stevens Lab.

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Mentzelia to Micro

Research did not cross my mind before coming to ACU because I didn’t enjoy labs in high school, so I wasn’t interested. However, my freshman year I had classes with several sophomores, who would often talk about the research they were doing. They made it sound intriguing and fun yet challenging, which was quite the opposite of my concept on research. As I walked by all the posters and different labs in the Foster Science Building (now known as Onstead Science), my curiosity grew, and, when Dr. (Josh) Brokaw sent an email for research applications, I signed up not really sure what I was getting myself in to. I was assigned to the Mentzelia research team, and I was extremely intimidated. I had no clue as to what Mentzelia was, which turned out to be a flowering plant, or the specific end goal for the research team. I showed up the first day of research completely clueless, but this is an aspect of research that I have grown to love. I started with little knowledge of my topic, but slowly developed skills and knowledge throughout the year. Most of the work that was done with the Mentzelia team was to run PCRs and gel electrophoreses because the end goal was to compare several Mentzelia species’ phylogenetic relationships and determine if there were any patterns of homoplasy (misleading similarities) in the DNA sequence data or morphology.  I realized that patience was a key characteristic to acquire during research.  As a team, we ran over 50 PCR reactions and gel electrophoreses with few positive results. It was frustrating, however, I was able to practice basic technique that has been valuable in my current research.

Hofsommer et al., 2016b

The end goal of any research is to be able to present your findings to an audience, and I had to give an oral presentation along with one of my team members. This was a hard task because most of our results were negative. Also, I did not believe that I had the knowledge to give a well-informed presentation, but with Dr. Brokaw’s guidance, we were able to develop a well-formed presentation. I still remember the question one of judges asked me, “Why is this research important?” I blanked. I was able to come up with a quick response, but I had not thought about the importance of the research that I was conducting before this moment.

IMGP0969

I may still not completely understand the impact this research with the Mentzelia team had on plant biology, but I learned so much during my first year of participating in research. I learned that consistency, patience, and determination are key characteristics that are developed during lab work. I also learned that I enjoy research. I began clueless, and, by the end of the year, I was able to give an oral presentation over the information. The journey of research is exciting. The end results are unknown and anything can happen. This is why I decided to continue research.

I am now working with Dr. (Diana) Flanagan on the identification of unknown bacterial isolates from Sorcerer’s Cave. At first it seemed like this semester of research was going to be similar to my previous experience because the gel electrophoreses was negative or as a team we had inconsistent results. We had to use different methods to isolate DNA in addition to running new PCR reactions and gel electrophoreses. But after changes to the methods, we had positive results that could be sequenced, and we were able to present our findings to the ACU Biology Research Club at the fall research poster session. This research has been exciting because we are working alongside Dr. (Jennifer) Huddleston’s research teams, who are testing for antibiotic resistance in the same unknown bacterial isolates. It has been a good semester of research so far, but as a team, we hope to identify more unknown bacterial isolates before the ACU Spring Research Festival and the Texas ASM (American Society for Microbiology) Meetings. I am so glad I decided to join a research team, because it has been a rewarding experience that I would have not had doing any other extracurricular activity.

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Monitoring Wildlife in the Southern Rolling Plains Using a Camera-Trap at Water Catchment Stations

In the spring semester of 2016 I joined in a research project, which was just starting to formulate, with another student. Other than classroom-based research and presentations, this was my first time being involved in a research project. This particular project is just in the preliminary stages as we’re collecting our first data. Eric Dolezalik, the other student involved, had recently studied water catchment systems in depth and decided he wanted to apply that knowledge for wildlife research. The Abilene area had seen a scarcity of water in recent years. Therefore, wildlife in the area have been spreading out to fulfill their water needs. This research project is designed to determine the various wildlife species that are present and what their population and density is in the Abilene area, while providing another water source collected by a catchment system and measuring its impact on wildlife distribution. So through the guidance of Dr. Josh Brokaw, we decided to conduct a trail camera survey with and without the presence of water sources on the 402 acres of the ACU Rhoden Farm of the Agricultural and Environmental Sciences Department in Hamby, Texas.

Rolling Plains

Location of Rhoden Farm in the Southern Rolling Plains

Grid

Camera Trap Grid

With our study site determined, we set up a rectangular grid using Universal Transverse Mercator (UTM) coordinates on Google Earth. The reason we used UTM was to eliminate any variation in distance between coordinates (like with longitude) from the curvature of the earth, but once we got in the field, we realized our handheld GPS device did not have the UTM option on it, so we had to convert our points to longitude and latitude. With the grid established, we positioned trail cameras at evenly spaced locations throughout the farm, and we eliminated any points that were too close to a large manmade object or a sizable body of water or would interfere with any normal operations of the Rhoden Farm.  We ended up with twenty locations in all.  At these locations, we mounted trail cameras on T-posts to capture any animals that wander by. We are still in the process of building water catchment systems with the guidance of professor Billy Kniffen, a water resource expert, so all the data being collected on the cameras now will be used to establish a baseline of the wildlife presence before we put these catchment systems in place.

Catchment

Incomplete Water Catchment System

The catchment systems are small shed-like structures that are only about 3 feet high, 3 feet wide, and 6 feet long. It has a slanted roof to direct all the collected water to a 55 gallon barrel where it will be stored and then will proceed to concrete watering station just in front of the camera. Once we get the catchment systems in place, only ten of the camera locations will have them, and the other ten will be considered a control of water absence for our data collection. This preliminary stage has been useful because, after the first week of data being collected, we realized that our cameras were too high to capture much of anything smaller than a coyote, so we decided to lower all the cameras to about two and half feet off the ground, in hopes of catching more animals on camera. Thus far, without the water catchment systems, we have only been able to identify a few wildlife animals that have wandered in front of our cameras. We have positively identified white-tailed deer, black-tailed jackrabbits, great-tailed grackles, coyotes, scissor-tailed flycatchers, and a few others. Though in the future and with access to a supplemental water source, we foresee capturing a lot more wildlife on camera where we can compile the data and determine the species population richness, density, and diversity and measure the impacts of the water-catchment system. I look forward to seeing which species we’ll get on cameras and to see how they react to the increased water access.

deer

White-Tailed Deer

rabbit

Black-Tailed Jackrabbit

grackle

Great-Tailed Grackles

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Phylogenetic Analyses with New Nuclear Markers for Thomasomys

As my first research project as a student at Abilene Christian University, I was a member of the AP5 research team. The goal of our research was to confirm and further develop a newly constructed phylogeny of rodents in the genus Thomasomys. A phylogeny is a hypothesis of the evolutionary history and the evolutionary relationships between species. The reason that it is important to test the previously constructed phylogenetic tree is because a phylogeny is always a hypothesis.  The more DNA that is tested, the more confident we can be of the likelihood of our hypothesis. A fully resolved tree (where the exact relationships of all of the species are described) is important because it can be used to understand the impact of historical events like the formation of the Andes on the diversification history (how species separated) of Thomasomys. The phylogeny that has already been constructed was found using mitochondrial DNA, and our research was attempting to test the mitochondrial phylogeny with nuclear DNA. The mitochondrial genome comes from organelles called mitochondria (inherited from the mother) and contains anywhere from 100 to 1000 copies per cell while nuclear DNA is found in the nucleus (inherited from both parents) and contains two copies per cell.

T.caudivarius

Thomasomys caudivarius

In order to collect DNA from the AP5 (intron 2 from the acid phosphatase type V gene) region, we conducted polymerase chain reaction (PCR) on DNA samples from species of Thomasomys with primers that we learned about from previous research on rodent phylogenies. PCR is used to amplify a piece of DNA until there are millions of copies of the DNA sequence. DNA polymerase is the enzyme that replicates the DNA, and primers are short strands of DNA that are used to facilitate DNA replication because the polymerase requires an existing strand of DNA to which it can bind the replicated DNA. However, these primers didn’t work very well with Thomasomys because they were designed for other rodent species, but we were able to amplify AP5 from a few of our samples. We used the sequence that we obtained to design three new sets of primers, and two of the three sets were successful at amplifying AP5 from most of our samples. After the DNA is amplified using PCR, the product of the PCR is then tested to see whether our targeted area was present with gel electrophoresis. Electrophoresis separates different sizes of DNA strands. If there is DNA of the correct size present after the gel, then we assume that the AP5 region is probably present, and this DNA will then be cleaned and sent off to be sequenced. This sequence is what is then used to estimate evolutionary relationships between species by looking at how the sequences vary from each other.

AP5 Network

Figure 1: Genotype network representative of the AP5 sequences collected. The segments in between each oval represent a different mutation. The black ovals represent the results from primers used initially, and the red ovals represent the results from primers designed in our own labs. Although the red samples are related in a pattern congruent with mitochondrial hypotheses, they differ by only a small number of mutations in AP5.

Sequences from the AP5 gene have been both easier to obtain and easier to interpret and edit after the change in primers.  The resulting sequences from our new primers resulted in a preliminary phylogeny that was compatible with phylogenies based on mitochondrial DNA. However, the AP5 gene did not help create a more resolved phylogeny due to the lack of variability in the AP5 marker compared to the mitochondrial markers and other nuclear markers like RAG1.  Since our project began, we have learned of more promising nuclear markers that we will focus on in the future.

Other than the lack of variability of the AP5 gene, there are other potential causes of the lack of success with AP5 such as: human error in running the PCR or gel electrophoresis, the primers, or the temperature of the PCR amplification. As my time as a researcher on the AP5 team progressed, the amount of successful AP5 PCRs detected by electrophoresis increased gradually. This lends me to believe that human error caused by inexperience may have played a small role in difficulty of obtaining results. My skills in conducting PCR and gel electrophoresis improved as a result of this research, but more importantly I gained experience in applying knowledge learned in class to the real world. As a recent transfer to ACU, being a part of ACU biology research was an incredible way to feel included at a new university. There is a sense of community within research at ACU where students help each other learn and grow in a comfortable environment, which is a unique aspect not found at every institution. Towards the end of the semester, we participated in the ACU Undergraduate Research Festival with a poster presentation. As part of this presentation ACU professors circled the room asking questions of the presenters in order to test their knowledge of the research. This was my first experience at a research conference, and, being new to research, I was extremely fearful. Although initially this was a difficult experience for me, by the end I was grateful that I was a part of it. Hearing the more experienced members of our team answer questions gave me a far better understanding of our research, and I will be able to use this experience in the future.

IMGP0873

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Phylogenetic Analyses of Species in Thomasomys (Rodentia: Cricetidae)

Picture for Blog Entry

My research experience with the biology department has certainly been a memorable part of my time here at ACU. Starting in January, I jumped in on a project of Dr. Lee’s, a portion of which he delegated to Dr. Brokaw – the continuous updating of an exhaustive taxonomic tree of a rodent genus living in Ecuador named Thomasomys. This genus of mammals living in Andes Mountains has undergone rapid adaptive radiation, occupying a variety of niches, differing in climate and elevation. Dr. Lee and Dr. Brokaw are attempting to piece together the evolutionary relationships among species within this genus.

Here are pictures of our phylogenetic trees from two different genes, cytochrome b and CO1 (both mitochondrial).

Published Image

This image includes DNA we sequenced in the spring of 2015 and was featured in a publication by Dr. Lee and Rachel Ritchie. Before getting included in publications, any DNA we sequence is submitted to GenBank and becomes accessible to researchers worldwide.

CO1 ML Tree With Bootstrap Values 27-Nov-15

This image is a phylogeny based on our latest sequencing of the CO1 gene.

These are our results after the meticulous processes of running PCRs, gel electrophoresis, DNA purification, DNA quantification by NanoDrop, and sequence editing and alignment. During my participation in this project, we added two new species to our trees, T. ucucha and T. rhoadsi, found out that T. cinnameus may actually consist of two different species, and elucidated the confusing geographic distributions of the closely related sister species, T. baeops and T. taczanowskii.

Although the principal goal of the project isn’t entirely fascinating to me nor directly relevant to my future career, the experience I’ve gained working in the lab is something that I can take with me for the rest of my life. Extracting DNA, using a thermal cycler during the PCR process, running an electrophoresis gel, purifying DNA, and editing sequences have all been extremely useful techniques that I’ve learned. It’s also been fun getting to work with fellow classmates, and I learned a lot from other students like Tina Johnson, who was Dr. Brokaw’s lab mentor in the spring. Also, presenting at the ACU Research Festival was a fantastic experience, even though it was a little nerve-wracking.

One of the difficulties that I initially came across was the fact that I had no idea what was going on scientifically when I first started. I didn’t even know what “PCR” stood for until the day before presenting at the festival [The polymerase chain reaction is used to replicate DNA]. Creating a “master mix” for the PCR and loading the gel are meticulous processes that require complete focus, and I made silly errors at first because I wasn’t careful enough. This semester, my group had to repeat two steps, PCR and gel electrophoresis, multiple times, and it felt a little rote at times. We made a lot more progress in the spring semester, probably because the goal of presenting at the ACU Research Festival was more imminent. I also learned that research doesn’t turn out the way you plan it a lot of times – things go wrong more often than expected, like the way our PCR’s have turned out this semester.

Doing research while taking classes such as Cell Biology, Biostatistics, and General Biology II is a great way to apply what you’ve learned in a hands-on way. It’s also a great way to get to know the professor that you’re working for and to have the opportunity to ask him or her questions outside of the classroom. I believe that doing research for the biology department at ACU is an experience you don’t want to miss.

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Reflections on 2013/2014

It’s hard to believe the school year has finally come to an end—and even harder to believe that I began working on my summer research project about a year ago (oh, how the time flies). At the beginning of the summer, we were designing primers, and now we’re in the process of identifying specimens using SNP haplotyping. While we haven’t made as much progress as we initially envisioned (due to technical difficulties, among other things), we still identified quite a few and have made quite a dent in this continuing project (which we look forward to working on more). If nothing else, analysis of our soil collections suggested edaphic specialization in Mentzelia monoensis, which was a really exciting result to share with the world.

This semester I was not able to spend as much time in the lab, but I did present a 15-minute oral presentation at 3 different conferences. In January, we (Dr. Brokaw, Christian Hofsommer, and I) presented our work in Austin at the conference for the Society for Integrative and Comparative Biology. It was scary presenting my work in front of an audience—but it helped me to realize that I am fully capable of presenting information to a scientific community. In March, I  (in addition to several other biology and English students) presented at Alpha Chi’s National Conference in St. Louis—which was quite different from the Evolution and the SICB conferences. This conference focused primarily on undergraduate research and had presentations in over 26 different categories! So, it was really a unique experience to be exposed to so many different disciplines. I shared my work to an audience (that included my wonderful friends!) during the Organismal and Ecological Biology section. Elisa Wyrick (who researches British Literature) and I won best presentations in our categories—which came as quite a surprise! Finally, I presented my work for the last time at the ACU Undergraduate Research Festival at the beginning of April. By the end, it felt like I had come a full circle–presenting my research to my teachers and peers. Additionally, we (Dr. Brokaw, Christian Hofsommer, and I) are working on a publication, so stay tuned for more on that!
Ultimately, I feel I learned quite a lot during this past year. Slowly (but surely), I’m morphing into a scientist: I’ve learned to think more critically, ask more questions, and take more risks. And, as I’ve mentioned before, I’ve learning that failure is part of the process. Things don’t always turn out the way we’d like them to—not to mention everyone messes up from time to time. I definitely didn’t envision the primers or the specimen from different herbaria to give us difficulties, but unfortunately they did, and we had to troubleshoot around them. Despite these challenges, I’ve developed more independence and confidence in my research abilities as well as myself. I never could have imagined I would be able to explain my ideas to a room full of people—yet here I am on the other side.

A year later and I have presented at 3 national conferences, conducted field work, experimented with new methodology (primer design, allele-specific PCR, etc.), and won an award for my presentation (this one I still can’t believe)—not to mention I made many new friends! It’s difficult to articulate really just how much I have benefitted from this entire process, and I am so grateful for the opportunities research has given me (and many others) during my time at ACU. Unfortunately, this summer I will not be continuing my M. monoensis work, but instead I will be researching molecular and cell biology at Pepperdine! It’s a bit intimidating to be moving into a new lab with new people, ideas, and methodology—but ultimately, I know my experiences at ACU have well prepared me for this new adventure. Also, during the fall I will be working as a research tutor—which I honestly couldn’t be more excited about. It will be truly awesome to teach other students about research and share my passion with them.

All of the ACU Alpha Chi members attending the conference in St. Louis!
All of the ACU Alpha Chi leadership attending the conference in St. Louis!
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Bat Research Class

I began research with Dr. Brokaw and Dr. Lee at the beginning of Fall 2013. They wanted to start an entirely new project with me, so a lot of the beginning stages were simply figuring out what to research. Our options were really between rats and bats that Dr. Lee had collected. So, we went down to the natural history collection and looked at what species Dr. Lee had collected a lot of. He had a lot of Mexican free-tailed bats, or Tadarida brasiliensis, that he had been wanting to do a project with, so we ended up designing an experiment around that. Since he had samples from both Ecuador and Texas, two geographically distinct areas, we decided to see if there was any genetic variation between the two populations. Dr. Brokaw taught me DNA extraction and PCR first. However, we had quite a bit of trouble getting a good PCR for the majority of the experiment. After months of not being able to figure out what I was doing wrong, Dr. Brokaw was finally able to confirm that some of the chemicals I had been using were degraded. Once we fixed that problem, my PCRs worked fine. We cleaned and nanodropped (measured the concentration) the DNA from the cytochrome b gene before sending it off to another lab to be sequenced. Somewhere along the line, I also acquired a couple of research partners on the project. However, we usually worked on different schedules and got back together to share results. At the beginning of the spring semester, I finally met my partner that I continued to work with, Lindsey Bloomer (our other partner is currently on study abroad). Another road block that we had involved one of the coolers in the lab breaking down several years ago. It turned out that many of our DNA samples from Texas got ruined when they had thawed, and we only had one sequenced sample from Texas. We had to push our project to the very last minute to fix this. I literally finished the last set of DNA extraction, cleaning, and nanodropping all in two weeks. Luckily, we got four more sequences from Texas thanks to samples from the natural history collection at Angelo State University. Towards the end of the project, we edited the sequences and constructed a haplotype network of the cytochrome b sequences from different samples, which gave us some very interesting results. The bats from North America were put into a completely different network than the bats from South America (at this point, I had pulled some additional sequences from GenBank to compare to our new samples). Overall, we ended up deciding that the Texan bats probably did not migrate to South America and vice versa because there were no shared or even similar haplotypes in both locations. We plan to continue this research next year with the focus on a different gene that mutates a bit slower to find similarities between to two groups. My favorite parts of the project were trying to extract DNA from a bat foot and being asked by Dr. Lee to look for a sick bat that someone had reported was lying on the ground by the library. I really enjoyed learning from my wonderful mentors, and I am really excited for next year.

Bloomer and Burt

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Bioremediation Research Course

Over the course of this past year I have been working with Dr. Brokaw on the effects of remaining hydrocarbons on vegetation following bioremediation of a crude oil spill. This project was renewed back in 2010 after nearly a decade of allowing revegetation to the testing sites. I was privileged enough to be allowed to come into this research at the beginning of the Fall 2013 semester to continue what the previous researchers had been working on. Dr. Brokaw and I began by testing control groups of a C25 hydrocarbon and a C36 hydrocarbon on the GCMS (Gas Chromatograph Mass Spectrometer) to get a better understanding of the unknown hydrocarbons that would be tested from the soil. We did this by taking known solutions of either C25 or C36 mixed with cyclohexane, and then we used the GCMS to measure them. After coming to the conclusion that the concentrations of C25 and C36 can be reliably measured and that they produced very similar results, we have moved on to begin re-measuring the soil samples that were collected a few years back that might have similar hydrocarbons in them. We ran into a problem just recently with the GCMS breaking down on us, but that will not be a problem for us to continue later in the summer. The amounts of hydrocarbons in the soil are measured by washing the soil with cyclohexane by use of Soxhlet extraction. I plan to begin this part of the research this summer in June after Dr. Brokaw and I travel back to the spill site to collect more samples. I am very excited and interested in the continuation and results that this research will bring.

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Being involved in this research has been such a blessing by the Biology Department, but mainly Dr. Brokaw. Having a mentor as patient and understanding as him has definitely made this experience all the more amazing. I think that the opportunity to participate in graduate research and build relationships with the professors here at ACU is unparalleled and is really the ACU difference.

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Aeromonas and Research Tutors

In the last post I was about tell everyone how I got a third degree burn from making enchiladas and (this is embarrassing because I do microbiology research I should know better) how it got infected and I was out of the lab for three days. Bacteria are no joke. Who knew a tiny burn could make you so sick. It felt like I had the flu. All I did was sleep and I could not use my entire arm because the pain was so bad. Don’t do that kids. At least now I have cool pictures of all the layers of my skin growing back and scarring so that Dr. Huddleston can show her Anatomy classes. Fast forward the summer and the fall semester (I really should have kept up with this blog, whoops) and here we are at my last semester at ACU.

This semester we are welcoming two more new people to the Huddleston research group. Welcome, Sally and Kristen! Now there is total of six people in our lab. This year of research may not be as exciting as last summer when we welcomed two new people to our lab group, the Huddlestons welcomed baby Luke, and I had my near death experience, but we plan on making big progress this semester. We are a lot more focused this semester thanks to the new research class. Five members of the Huddleston lab are now also taking a research class for upper-level biology credit. This allows us to learn new techniques and have lots of a time to work in the lab. We will be attending and presenting at the Undergraduate Research Festival at ACU and at the Texas ASM conference in New Braunfels later in April.

Right now we are working on a way to get our products AB and C to form ABC using the SOE PCR technique. We have made new reagents, ran the DNA out on a gel to make sure it had not degraded, tweaked the PCR protocol, and checked the primers. Next, we plan to increase the concentration of template added to the PCR mix to see if that has any positive outcomes. Once we get the ABC product made we can incorporate it into our Aeromonas cells and get moving on research. You have to be a really patient and meticulous person to get through research sometimes. The roadblocks are discouraging sometimes, but I really want to make a lot a progress this semester. This is all the time I have left!

I plan on working on my complementation vector this week- Sing Song willing- if it doesn’t snow again like it did last week when I was supposed to work on it. The gene of interest has already been ligated into the vector. I just need to transform the vector into E.coli cells to screen them for the recombinant plasmids.

ACU Biology Research Tutors Kathryn Davis and Madeline Peterson
ACU Biology Research Tutors Kathryn Davis and Madeline Peterson

Another fun thing about this semester is that Dr. Brokaw asked Madeline and I if we wanted to be the first research tutors for the biology department. Every Wednesday and Friday one of us is in the lab to help with lab upkeep and teaching new student researchers the basics of biology research. We teach them how to run PCRs, DNA extractions, and how to run EtBr or SYBR gels. The necessity of lab tutors shows just how much the biology research group has grown in the past couple of years. Yay, biology!

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Mentzelia and DNA

During the last year, I’ve been doing research with Dr. Brokaw on the genetics of species in the genus Mentzelia. This research involves sequencing the DNA of these plants and looking for similarities and differences between multiple species in this genus in order to see if the regions we’re using are a definitive indicator of a difference between the species. In addition, we’re using it to compare a few species in particular, with a special focus on Mentzelia monoensis. This species, thus far, has only been found in one region in Mono county, California. Because the traditional means of differentiating it from other species, it’s seeds, is not always feasible depending on the season and the presence of seeds in a particular sample, it’s important to be able to identify it through alternative means. To this end, we are comparing the chloroplast DNA in Mentzelia monoensis to species that are difficult to differentiate by sight alone.

christian
My time in the lab has primarily been dedicated to the extraction of the DNA and interpreting the results of this DNA. The first step in the process is the collecting of a sample from a pressed plant, and grinding the plant matter so that the plant cell proteins don’t interfere with the DNA extraction process. This is probably the most tedious step in the process, especially when working with many samples at once. Often the pressed samples are difficult to get any leaves from, and moving the leaves takes some precision. The grinding itself can be pretty tiring on the fingers, as the leaf samples are rather small and therefore necessitate using a very small pestle to grind them, which can strain one’s fingers after a while. Even though this step can be boring, it’s also very interesting. The samples we use often are from very different geographic regions, given to us by herbariums across the area that we’re studying. In addition, the samples are very frequently older than myself; of the oldest samples we discovered was over 100 years old!

The rest of the process often goes much smoother, with enough to do to remain interesting. After extracting the DNA by grinding it, we have to purify the DNA and then amplify it through a polymerase chain reaction that essentially copies the DNA several times. We then send the DNA off to a lab for them to sequence, and we receive the results, correct any errors, and compare them to previous samples. The entire process has been fairly exciting. It’s been great to see our results accumulating over the last six months and supporting our hypotheses about the plants we’re studying. I’ve learned quite a bit about the research process, especially how to avoid mistakes (it can be rather difficult to figure out what a sample is when you rub half the label off!), and I’ve gotten a lot of valuable lab experience working with machines and processes that I might encounter later in my life. While we haven’t been able to get many results recently because we haven’t quite mastered our new extraction process, I’m looking forward to continuing to proceed and eventually get more done. Our results have thus far been interesting, and I’m excited to see where they go in the future.

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