Fun, Physics, and Sunburn

Fun, Physics, and Sunburn

Summer is a time for fun, physics, and sunburn. The summer I have had here in Gettysburg, PA has encompassed all three of those perfect aspects. My usual weekday starts at 6:30AM going to the gym. This has been a practice that I established during the school year and have not broken yet. My roommates, did I mention I lived with 8 girls?, even started joining me to go over and work out together.

After the gym I come home, push through the exhaustion and soreness, and make myself breakfast. In true Gettysburg fashion, I don’t just settle for pop tarts for breakfast. No, I make myself eggs-over-easy with peppers and onions, and occasionally bacon. Mmmmm, bacon. What makes my breakfast even more special is that I purchase the eggs from a local farmers market that is held every Wednesday. The eggs are farm fresh, usually gathered the day before purchase.

After my morning routine, I make my way over to work. This summer I had the pleasure of working with Prof. Bret Crawford as my mentor. The research field that we focused on is nuclear physics; specifically the weak nuclear force. This was done by working with a computer simulation of an experiment that is occurring at the National Institute of Science and Technology(NIST). The experiment at NIST is composed of a slow neutron beam that is used to study neutron-neutron weak interaction. This is done by first polarizing the neutron beam by using a super mirror polarizer. This aligns all of the neutrons along the y-axis. The beam then enters a target chamber that is filled with liquid He. Through the weak interaction between the neutron and the He nuclei, the helicity states of the polarized neutrons begin to shift out of phase due to the parity violation of the weak force. This difference in phase, causes the neutrons to almost corkscrew in their rotation. By measuring this parity-violating corkscrew, insight can be obtained into light nuclei interactions and the strength of the weak interaction.

My specific contribution to the project is that I created another aspect of the code that simulates the last aspect of the experiment, an ion chamber. The ion chamber is filled with gaseous 3He that collects the neutrons as it goes through it. The chamber is split into four sections along the beam direction; by breaking the chamber into sections, the neutrons can be collected by energy because the higher the energy of the neutron, the deeper the neutron will travel into the detector. This part of the simulation can, and is, being used to determine how much 3He the ion chamber should be filled with. The goal would be to have equal detection in all four sections while having minimal loss of neutrons out the back and sides of the detector. Also, the simulation can be used to study the spectra of the neutrons that are collected in each section by using the computer program PAW. By studying the the theoretical spectra of each section, more can be learned about the energy spectrum of the beam itself. So basically, using this simulation we can run the entire experiment with 10^6 neutrons in 10 seconds, never leave my chair, and learn so much about how changing one parameter of the experiment may change the actual experiment.

 

Spectra of collected neutrons in the four sections of ion chamber

Figure 1. Spectra of collected neutrons in the four sections of the Ion Chamber

 

After work, I tend to go home and have a small snack to help refresh me from my day. I then go over to the college’s tennis courts and play tennis with a group of friends that are also on campus. There is nothing better than to go outside and be active after a long day of sitting in front of a computer. This summer I learned how to serve overhand and I “obtained” an acceptable backhand. After tennis I usually end my day by cooking dinner. My favorite way to make dinner is grilling it. Usually chicken breasts, but when I’m feeling a bit more excitement in my life, I’ll buy and grill a New York strip steak. Corn on the cob, cooked on the grill is probably my favorite side.

On the weekends, the Gettysburg area has proven to have plenty to do. Some weekends are spent just around campus or exploring the battlefields. Others are spent exploring the surrounding area. A day trip to a lake in Pine Grove Furnace State park showed me that you do not need to be by a beach to bury your feet in sand and burn in the sun. All of the fun, and everything that I have learned has made this summer one that I will never forget.

Diggin’ through the desert for a phage with no name…

Diggin’ through the desert for a phage with no name…

With the stars still visible in the morning sky, we lifted our luggage into a van bound for BWI, our heads leaking the tailings of a Genetics final finished hours prior.  Our 7:30 flight to Las Vegas was packed with young adults and old couples, all chatting about what four-star hotels they were staying in and which casinos have the best slots.  We were Sin City bound, and we were likely the only people on the flight not heading straight for the strip.  In the eight days that followed, we, three students and our Chaco-clad professor, gallivanted across the American Southwest, sampling soil and salsas on the road from Vegas to Tucson.

 

Scientists of varied backgrounds have followed a similar trail for decades, observing and recording the unique geology and plant life of the region.  We admired the sun-baked landscapes and watched our footing among the less-than friendly flora, but our focus was drawn to the desert dirt, home to many members of the bacterial genus Bacillus.  The bacteria are merely the hosts for the primary reason of our trip, namely the tiny and plentiful viruses known as bacteriophages.

We are phage hunters.  And if the relevant literature gives any indication, our prey surrounds us.  Phage are in the water and soil, carried by the clouds and nestled in termite guts.  Where a prokaryote goes, you can bet its parasitic partner is not far behind.  With great abundance comes great variety, particularly when it comes to the genomes of these marvelous microfauna.  Our research as a lab deals dually with characterization of novel phages and processing of the existing stores of data.  While there are practical applications to our work, the whole field of viral biology is driven by pure scientific inquiry – how did this immense ecological variety arise? What genes account for the different infectious properties of these bacteriophage?  Though we’ve only scratched the surface, answering these questions will give insight to the evolutionary history of the Earth’s most plentiful organisms.

#dogreatwork

My name is Katherine Boas, I am a junior Biochemistry and Molecular Biology major and I work in the Krukonis/Delesalle lab, where we study bacteriophage ecology. Due to the miniscule size of bacteriophages just a spoonful of soil allows us to glimpse into the uncharted world of the phage. Bacteriophages are pivotal in the evolution of bacteria as well as the genetic changes that occur in soil, vegetation, and the oceans. Of these viruses that infect bacterial cells, we are specifically studying phages which infect Bacillus subtilis, a bacterium commonly found in soils where stress and starvation are common, such as the American southwest. Nine strains of Bacillus subtilis will be used to examine the host range of the bacteriophage in our soil samples. The DNA of four already sequenced Bacillus subtilis bacteriophages will then be used to probe the naturally occurring bacteriophage from the samples. What does it mean to probe the sample? Great question; a Roche DIG High Prime DNA labeling and Detection kit is used. Essentially, DIG (digoxigenin) is added to the genome of a phage through random priming and a probe is synthesized. DNA of phage to be probed is linked to a nylon membrane and then washed with various solutions one of which is the denatured probe. The DNA of the probe binds to complementary DNA linked to the membrane. Detection of the probe is performed using anti-DIG antibodies that only bind to DIG. The similarity of the phage DNA on the membrane to the DNA of the probe can then be visualized using CSPD (Chemiluminescent substrate for alkaline phosphatase). This technique is perfect for the assessment of phage diversity in the soil samples. By looking at the diversity of naturally occurring bacteriophage we can better understand how they interact with their hosts and thus understand the structure of these  communities. My focus this summer was to develop and refine the probing protocol.

Do ya DIG it?

Membrane Hybridization visualization

 

I am Albert Vill, a junior Biochemistry and Molecular Biology major working with Professors Krukonis and Delesalle.  While I have played a part in our lab’s Bacillus phage project, my personal work involves phages of Mycobacterium smegmatis (M. smeg).  So far, all characterized viruses of M. smeg contain double-stranded DNA (dsDNA) as their genetic material.  However, some bacterial hosts with known DNA phage have been found to have RNA-based viruses as well (e.g. DNA phage λ and RNA phage MS2 both infect E. coli).  We hypothesized that M. smeg has RNA phages, but that such viruses are less prolific than their dsDNA counterparts.  In an attempt to isolate an RNA virus from soil, I have modified an enrichment protocol to include hydroxyurea, a ribonucleotide reductase inhibitor used to prevent DNA replication without interfering with the synthesis of ribonucleic acid or proteins.  Essentially, the DNA phage present in the enrichment solution would not be able to reproduce, giving any RNA phage present a chance to proliferate.  Once a phage has been isolated and a high-concentration stock created – at least 10^8 viral particles per milliliter – I use electron microscopy as a screening process before continuing with additional tests.  A normal light microscope, sufficient for viewing animal and plant cells, is not powerful enough to resolve individual phage.  The average mycobacteriophage is only a few hundred nanometers from head to tail, meaning that 1500 phage could be arranged end to end around the circumference of a human hair.  Under the microscope, DNA phage have capsids (sphere-like structures containing the genetic material) with long, thin tails, whereas RNA phage consist only of capsids.  The preparation of the microscope samples is difficult, given that the tails can be easily “knocked off” of DNA phage, leading to false positives and necessitating procedural prudence and multiple samples.  If this isolation procedure is fruitful, further experiments will be done in coming semesters to extract RNA and reverse-transcribed DNA from the putative RNA-phage sample.

IMG_0006

DNA mycobacteriophage enveloped in phosphotungstic acid stain at 30000x magnification

 

I’m Brianne Tomko, also a junior Biochemistry and Molecular Biology major in the Krukonis/Delesalle lab.  This summer, in addition to collecting and working with the desert soil samples, I’ve been working with Bacillus phages isolated by Kendra Hayden ’12 in 2011 from soil collected in Tucson, AZ.  Kendra isolated 10 phages, whose genomes have all been sequenced and are in various stages of assembly.  In particular, I am working on annotating and characterizing one of these phages, K7.  Its relatively short (46,270bp), circular genome has been fully sequenced and assembled.  This is done by using the assembly program Newbler. It takes the tens of thousands of short sequences (reads) produced by the sequencing process and aligns them to put like sequences together and create longer contiguous sequences (contigs) that can be viewed in another program, Consed.

Newbler

Alignment of reads in Newbler

Sometimes the raw data will be assembled into one sequence, but other times, Consed will be used to put contigs together.  With Consed, you can look for similarities between the contigs to join them together into one sequence among other things.

Consed

Assembly View in Consed – the orange and black lines signify sequence similarity

Currently, I am annotating the genome to identify potential genes and their putative functions.  This is done by using various computer programs and databases to compare the genome in question to previously characterized and well-studied genomes.  K7 is closely related to another previously annotated Bacillus phage, SPP1, so I have also spent time studying SPP1 and comparing the two phages. These phages are also similar to another phage, PM1, which was sequenced in 2013.

Our next step is to grow more K7 to get mass spectrometry data.  The mass spectrometer separates proteins based on size and charge, and the output gives the mass and relative abundance of each protein in the sample.  This can also give information on the structure and function of the proteins. Phage genomes are very small, and from studying groups of similar phages, we know that the genes are tightly packed together, with very little unused space.  The structural genes in bacteriophages are pretty well studied and can be easy to identify, but the great majority of genes do not have easily detectable or well-studied functions, so there is a lot left to learn about these phages and their life cycles.

 

 

Exploring the Dynamics of Galaxy Cluster Mergers

We are Tessa Thorsen and Andre Hinds, rising Juniors in the Physics Department at Gettysburg College. This summer we worked with Dr. Johnson analyzing galaxy cluster mergers. Galaxy clusters are gravitationally bound groupings of large numbers of galaxies and smaller subgroups, leading to the presence of complex substructure within the cluster.

ngc1275-perseus-cluster

Abell 426 – Perseus Galaxy Cluster. Source: universetoday.com

There are many theories on how these clusters form, so looking at the merger process can give us insight as to which of these theories might be most applicable. Galaxies, and the clusters they form, are actually extremely sparse, so during a merger the only collisional matter is the interstellar gas. For our research we are not looking at this gas. Rather, we are examining what happens to the non-collisional matter that makes up the galaxies. Our goal is to determine statistical techniques for deciding which galaxies belong to which clusters, finding substructure in the clusters, and describing the dynamics of the clusters at different stages in the merger.

The video below is a visualization of simulated galaxy cluster mergers. The first 50 seconds of the video show how particles in the two different clusters, yellow and blue, interact throughout the merger. The rest of the video shows the collisional gas during the merger, which is interesting, but not our focus.

We have been looking at the same data presented in the video, which was created by Dr. Zuhone, one of Dr. Johnson’s collaborators at the Goddard Space Flight Center. The data contains nine different simulations, each with different initial parameters representing the mass ratios of the clusters and the angle of approach between the two clusters. Each simulation contains 100,000 particles, which are representative of the dark matter present in the clusters.

We are looking at simulated data rather than observational data because it allows us to manipulate time and space in a way that is impossible when performing observations through a telescope. With the simulated data we can see positional and velocity data in three dimensions, we can observe the entire merger throughout periods of hundreds of millions of years, and we have a priori knowledge of which galaxies belong to which clusters. This means that we have a better idea of the accuracy of any tests we perform.

From here we have split the project up into two parts. The first part is concerned with determining cluster membership of galaxies throughout the merger. We have attempted to apply a partitioning algorithm developed by mathematicians and other researchers in this field, and have compared the results to the known cluster distributions. It is fairly simple to determine membership at the beginning of the merger, but as the clusters begin to coalesce it is harder to distinguish between them. The algorithm requires fairly exact estimates of  the average velocity and position of each cluster, so we need to determine a better method for approximating these measurements, without prior knowledge.

The second part focuses on describing how the clusters behave throughout the merger. We are projecting the three dimensional data we have onto a plane, representing the plane of the sky, because it allows us to see the simulated data as we would see observational data. From there, we have looked at the change in the central position and scale of the galaxies throughout the merger, as well as the velocity dispersion. The velocity dispersion is a measure of the difference in velocity of the particles throughout the cluster, and is a very helpful statistic for comparing the dynamics of clusters. We have found that, as the clusters approach each other, their velocity dispersions increase dramatically, as particles begin to feel more intense gravitational attraction, and then flatten out as the merger progresses.

Graph of velocity dispersion and position of two clusters undergoing a merger.

Graph of velocity dispersion and position of two clusters undergoing a merger.

Studying galaxy clusters and their substructure can illuminate many of the mysteries behind the formation of our universe. Substructure can tell us how quickly our universe was and is expanding, how matter was initially distributed throughout space, and even how old our universe is. Much research has been done on this subject, and many of these things are becoming increasingly well known, but research such as ours comes at it from a different angle, and can provide a unique perspective.

Research Expedition to Nicaragua

We are Morgan Panzer and Ellen Petley, both rising senior Biology majors, who are working with Dr. István Urcuyo over the summer. His area of research focuses on the biodiversity of marine invertebrates, mainly of the phylum Mollusca, on the Pacific Coast of Nicaragua. Our work is separated into two main sections. At first, we worked on the sorting, organization and species identification of samples collected during previous research expeditions to Nicaragua (now housed in the laboratory at Gettysburg College). Currently, we are on our 3-week research trip in Nicaragua, and while in the field we perform collection of samples and also carry out water chemistry analysis of both phosphates and nitrates.

urcuyo 1

Our Lab Team after climbing to the top of Masaya Volcano dormant crater.

 

Most of our field locations are located at least two or more hours away from our home-based (an apartment in Managua), so a lot of our days consist of lots of travel in a very cramped rental truck. Generally, our field research teams have consisted of Morgan, Ellen, Dr. Urcuyo, Janina Urcuyo (malacologist), and Luis Canda (ecologist). The study sites that we have visited so far were chosen because Dr. Urcuyo and Luis deployed a thermistor (an underwater digital thermometer recorder) each summer on the ocean floor to collect ocean water temperature data every half hour for the entire duration of the deployment. This temperature data is an important characteristic, and indicator, of near-shore currents that affect the distribution and presence of marine species that Dr. Urcuyo is collecting in Nicaragua’s pacific coast. To complete this task requires a scuba diving expedition and a boat ride with a random local fisherman.

Part of our field work focuses on collecting samples to add to Dr. Urcuyo’s growing biodiversity collection and to add to the collection at the Malacology Museum at the UCA (University of Central America) in Managua. When we visit sites, we generally wade in tidal pools at low tide (when the most organisms and tidal pools are exposed), or we walk along the sandy beach to look for shells that are the least worn and the least broken for better quality of identification. Sometimes, low tide is at midnight and sometimes it’s in the middle of the day, but we have to be ready to look for samples regardless of the time or amount of sunlight. When we go out at night, we work with headlamps, and during the day, we work with plenty of sunscreen. Because the current mollusk collections in both the UCA’s lab and Dr. Urcuyo’s labs are fairly extensive, we have been generally looking for organisms that do not look familiar to us or for those that are especially nice samples. Samples without any live tissue within them (“dry” samples) are collected in a bag, while those that do contain living tissue (“wet” samples) are placed in a container and preserved in alcohol (70% ethanol and 30% water). From there, we label the bags and containers with the location name and date, and then take them back with us to be identified later on either at the malacology lab or our lab in Gettysburg College.

urcuyo 2

Looking for marine invertebrates in the tidal pools of Masachapa, Nicaragua.

While at the Malacology Museum in the UCA, we generally spend our time identifying the organisms that we collected in the field. For the “dry” samples, we place them all on a tray and rinse them of dirt and sand with freshwater. We then typically organize them based on morphological similarity so that those that we think are closely related are grouped together. After that, we use our lab mollusk identification reference book, Seashells of Tropical West America:Marine Mollusks from Baja California to Peru, which we jokingly refer to as the “The Mollusk Bible”. A very dedicated female Malacologist named Dr. Martha Keen spent most of her life classifying species of mollusks, and she wrote a book describing most known species of mollusk from the Eastern Pacific at that time. Each species description includes a paragraph describing the shape, coloration and other necessary minutia morphological characteristics (such as the number of ridges on the shell with very many other intricate details about the organism) often accompanied by a picture of the species. Each species of mollusk in each taxonomic Class is assigned a number, deemed the “Keen number”, which we use as a quick identification tool. Oftentimes, two species are nearly indistinguishable, so we use the collections at our lab in Gettysburg, the reference collection at the Malacology Museum at the UCA or the expertise of the biologists in our team to help us figure out the correct species identification. Sometimes, even they don’t know for sure which species the sample belongs to, so we set those samples aside to be taken with us on a future trip to the Smithsonian Museum for further consultation with experts and their reference collections.

urcuyo 3

The gastropod Macrocypraea cervinetta on the rocks at Chacocente, Nicaragua.

 

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After a long day of mollusk identification at the Malacology Museum laboratory in the UCA…

Paying close attention to small details carries over into the other portion of our field work responsibilities: the water chemistry aspect of our research. At each location we travel to, nitrate and phosphate levels are measured numerous times to add to previous collected water quality data. Using a field Colorimeter (Model 850, Hach Company), prepackaged chemical reagent samples and two glass vials, we follow the pre-set protocol for nitrate and phosphate respectively. Unfortunately for us this makes the curious fishermen even more curious about the two gringas (Spanish slang for American) working with strange apparatus and chemicals on his boat.

urcuyo 5

Ellen doing water chemistry, on her lap is the instruction booklet and field spectrophotometer. Photo by: Janina Urcuyo

When we aren’t in the boat rocking back and forth, trying to get a meniscus at precisely 10 mL with the waves crashing into us, Nicaragua offers a lot of relaxation. Overcoming the language barrier by talking to local street cats, dogs, and birds is really going well, as they seem to always understand our Spanish. As for actual people, not so much. Trying new cuisine is probably the hardest part of this job, as there are so many new fruit juices, infamous dishes and desserts to try. Each day is a new adventure, including having our truck broken into and the robbery of personal items, peeing in the woods in the dark, and getting rear-ended by stray motorcyclist (but no worries, our team is safe and sound, plus the motorcyclist only received two very minor scrapes. We also get to see some pretty cool scientific stuff, too. During our journeys, we’ve found baby octopuses, a mama Olive Ridley’s turtle laying eggs on the beach, some newly-hatched sea turtles, some mating sea turtles, owls, eels, electrocuted kinkajou, howling monkeys, and lots of military men with AK47s protecting the entry and exits to the natural reserves. The X-Sig program has given us the experience of a lifetime, SCIENCE RULES!

Electrocuted Kinkajou, Photo by: Janina Urcuyo

Electrocuted Kinkajou, Photo by: Janina Urcuyo

Some CSI business after our truck got broken into, photo by: Janina Urcuyo

Some CSI business after our truck got broken into, photo by: Janina Urcuyo

Playing with baby Olive Ridley (Lepidochelys olivacea) sea turtles! Photo by: Janina Urcuyo

Playing with baby Olive Ridley (Lepidochelys olivacea) sea turtles! Photo by: Janina Urcuyo

Working hard trying new foods (aka stuffing our faces whenever we can).

Working hard trying new foods (aka stuffing our faces whenever we can).

On top of Volcano Masaya. Thanks, HHMI!

On top of Volcano Masaya. Thanks, HHMI!

 

Sweet Tea and Symbiosis – Exploring Kombucha’s Detoxifying Effects

Hi! My name is Stacey Heaver and I am a senior BMB major spending my second summer in the X-SIG program with Drs. Krukonis and Delesalle. Last summer I worked on a bacteriophage bioinformatics project, but this summer I decided to mix it up and start on a new project that I’ll continue into the upcoming year – studying kombucha!

What’s kombucha? It’s sweet tea that’s been fermented by a symbiotic culture of bacteria and yeasts (SCOBY) into a slightly vinegary and effervescent drink. It’s thought to have originated in the Chinese Tsin dynasty in 212 BC and has been touted since by cultures worldwide as a health tonic – doing everything from preventing or curing cancer to treating arthritis and easing symptoms of depressive or anxiety disorders. Of course, we want scientific evidence before we can accept any of those claims!

Today, you can stop by the Gettysburg Giant and buy a commercial version of kombucha – and across the country, dozens of artisanal kombucha brewers are popping up. Each of these companies uses a different community of bacteria and yeasts to ferment their tea. While Acetobacter and Gluconacetobacter bacteria and Schizosaccharomyces and Saccharomyces yeasts are known to predominate in kombucha, there is no standardization of species in different fermentations – and many will have small amounts of very unique strains. Because the proposed health benefits of kombucha are expected to result from the metabolic products of the species present within it, variations in kombucha composition can change any expected health outcomes from drinking the tea. Knowing what specific strains make the healthiest kombucha would be a great asset.

kombucharainbow

Newly inoculated black, rooibos, and green tea beginning fermentation in the flow hood. The whitish solid seen floating on the top is the SCOBY – it’s a thick biofilm with cellulose scaffolding. Cutting into it, it has the same texture as raw chicken.

 

newSCOBY

A newly forming SCOBY.

As sweet tea ferments after inoculation with a kombucha SCOBY and starter tea, the first major metabolite produced is ethanol. As the ethanol concentration builds, the acetic acid bacteria grow more active, increasing the drink’s acidity and giving it a vinegar kick. The growing acidity helps make the tea broth inhospitable to a number of pathogenic bacteria that might otherwise threaten to overtake the fermentation. Lactic acid and glucuronic acid can also be produced in smaller amounts, the latter being involved in the body’s detoxification system.

Glucuronates are very polar, and by binding to toxins they solubilize and target them for expulsion from the body. They can bind both to toxins produced in the body, like endogenous reactive metabolites or the breakdown products from heme catabolism, or to external toxins introduced via environment or diet. The body also takes advantage of their solubilizing nature to send chemicals to their target tissues – like steroid hormones, fat-soluble vitamins, essential unsaturated fatty acids, and dietary polyphenols. We don’t have to produce brand-new glucuronates each time. Instead, we have an enzyme called β-glucuronidase widespread throughout our bodies, produced both by our own cells and the bacteria that inhabit it. This enzyme catalyzes the dissociation of the glucuronate moiety off of whatever compound it bound to.

B-glucuronidase reaction

Diagram via BRENDA database. The left compound is the bound glucuronate; β-glucuronidase catalyzes its dissociation and frees it to bind again.

 

When β-glucuronidase frees a toxin instead of a harmless compound, these toxins can interact negatively with the surrounding cells. In patients with colon cancer, the enzyme has been found to be 12 times more expressed in feces than in healthy patients. In patients with bladder cancer, β-glucuronidase has been found to be overexpressed in urine. However, the enzyme’s presence can also be used therapeutically, with glucuronide prodrugs targeted to tumor sites for activation and expression via the cleaving action of β-glucuronidase.

Because our gastrointestinal tracts are so frequently exposed to potential carcinogens (think heterocyclic amines in the black crust on a grilled steak), it seems that consuming these in tandem with a dose of glucuronic acid ready to bind them up would be a smart thing to do. But kombucha appears to take that even a step further, producing a dynamic duo of compounds – in addition to glucuronic acid, some kombucha samples also produce D-saccharic acid 1,4-lactone (DSL), which is a competitive inhibitor against β-glucuronidase.

DSL

DSL, a competitive inhibitor against β-glucuronidase. Structure via BRENDA database.

In my project, I am exploring how to change kombucha production conditions to maximize DSL content. The bacteria and yeasts present in each fermentation produce a complex interacting network of metabolites depending on which initial substrates they have to work with, and we’re able to change the initial type of tea or sweetener or ferment with an entirely new media, often with a unique bacterial population all its own (think kvass, kefir, wine, and beer). Kombucha is also traditionally fermented with an 8-14 day initial aerobic ferment followed by anaerobic conditions for 1-3 days, during which the acetic acid bacteria shut down and the tea becomes carbonated. Often different extracts are added for this secondary ferment, and any of these additions may have the power to change the DSL content of the final product.

Besides comparing different media for fermentation, I’m also looking at enhancing DSL production through the addition of the bacteria Gluconacetobacter saccharivorans. This bacteria is genetically closely related to Gluconacetobacter A4, which has been shown to produce large amounts of DSL. After collecting kombucha samples at different time points and in different cultures, I can filter sterilize the samples to remove any bacteria and yeasts but keep their metabolites. Then, by inoculating with G. saccharivorans, I will be able to observe how interactions with the previously made metabolites affect DSL production. By first sterilizing the samples, I will be able to see more specifically the effect this bacteria has without worrying about the unknown identities of the bacteria and yeasts in the different initial cultures. A large number of different starter samples will offer a level of reproducibility despite the unstandardized microbial composition of each culture.

My largest struggle this summer has been reliably quantifying the amount of DSL in each sample, learning how to use the capillary electrophoresis machine, and optimizing a protocol specific to this compound. I’m currently able to tell when I have more or less DSL, but I haven’t yet minimized the variation between runs of the same samples to have reliable enough data. I am grateful that I will have the next two semesters to continue working out the kinks and collecting data!

7_7_14_DSL_standards

An example of three overlayed runs of DSL standards of known amounts on the high performance capillary electrophoresis machine. The largest peak around 17 min in the top run represents the larger quantity of DSL in the sample, compared to the smaller peak when the compound is more dilute or the absence of a peak at the same timepoint when no compound in injected (bottom run).

 

I feel so fortunate to be able to spend my summer studying something as fascinating as kombucha. Our microbiome and its interactions with our environment are so wonderfully interesting. Plus, you can make this out of kombucha SCOBYs:BioDenim_jacket

image source

WHAT. tell me science isn’t awesome.

Learn more about wearing biofilms!

Nano Nano

Have you ever seen a nanoparticle? You probably haven’t ‘SEEN’ one since you could fit about 600 nanoparticles within the diameter of a single human hair, but you’d be surprised to learn that nanoparticles are everywhere. There are two types of nanoparticles, naturally-occurring and manmade. Naturally-occurring nanoparticles come in the form of volcanic ash and sea spray; manmade nanoparticles are formed as byproducts of car exhaust and mining, can be used in medicine, and are made in labs like ours. This summer the Nano lab incorporates three different projects, all dealing with a different aspect of gold nanoparticles: medicine, toxicity, and fundamental properties.

Why Gold Nanoparticles?

So why did we choose to work with nanoparticles, and more specifically, why gold nanoparticles? Well, nanoparticles are interesting to study because they exhibit properties that are very different from their bulk substances. For instance, a gold necklace will react and respond differently to physical and chemical changes than a gold nanoparticle will. One of the easiest differences to observe is the change in the way nanoparticles absorb light. Bulk gold all looks, well, gold. However, as the size of a nanoparticle changes, so does the way it responds to light.

Spheres to Long Rods

Nanospheres in solution appear red (far left), while long nanorods appear brown (far right), and short nanorods appear green (middle).

This same difference in properties can be seen in nanoparticles of other metals, such as silver or iron. We work specifically with gold because we have the ability to synthesize them in our very own lab using the process shown below. The ability to synthesize the nanoparticles in lab allows us to choose what size (and therefore, color) of particle we would like to make. We simply vary the amount of silver added in the scheme outlined below, and the size changes. The smallest amount of silver produces spheres, and as the silver is increased, the length of rods increases as well.  Making the nanoparticles in our own lab also ensures that we have the particles we want, when we want them, which means no waiting for shipments of particles to arrive before our research can take place.

Outline for the seed-mediated growth of gold nanoparticles

An outline for the seed-mediated growth of gold nanoparticles we use in our lab.

Who Are We and What Do We Do?

My name is Ida DiMucci. I am a rising senior Chemistry major and have done research in Dr. Thompson’s lab for two summers and one school semester in between. I am also an active member of Residence Life and will be the Residence Coordinator for Paul Hall this upcoming year. In addition to research, I also act as a Peer Science Mentor for the general chemistry classes and am currently the president of the Chemistry club on campus known as Sceptical Chymists. In my free time, you will find me climbing at the rock wall in the Den, running on the battlefields, or at Mr. G’s getting ice cream. After graduation, I plan to attend graduate school and receive a Ph. D in Chemistry, eventually returning to a small school like Gettysburg and becoming a professor with adorable children like Professor Frey.

In the Lab

While the study of nanoparticles may seem like new up-and-coming research, they have actually been around since the creation of stained glass windows during the medieval period.

The different colors in stained glass are caused by differences in nanoparticle size and shape.

The different colors in stained glass are caused by differences in nanoparticle size and shape.

Obviously, in the last 2000 years, many advances in the study of nanoparticles have been made, from using them in facial cream to using them in photothermal therapy to destroy cancer cells. My work with gold nanoparticles follows this trend and utilizes their unique structure and optical properties for the potential of drug delivery. While a nanoparticle may seem small when compared to a human hair, when thinking about them from a chemistry point of view they are actually gigantic. As seen in the picture below, about 30 benzene rings could fit across the diameter of one nanosphere.

A typical gold nanosphere has a diameter 30 times that of a benzene ring.

A typical gold nanosphere has a diameter 30 times that of a benzene ring.

This raises the question: Could small molecule (in particular, medicinal molecules) be somehow attached to a nanoparticle, allowing the particle to act as a drug delivery device in the body?  My work involves preliminary studies on what types of molecules could be attached to gold nanoparticles and how that attachment would take place. When synthesis is completed, the gold nanoparticle core is surrounded by a layer of CTAB, which is a surfactant or soapy molecule which consists of a long hydrophobic tail and a hydrophilic head. As shown below, this hydrophobic tail makes it possible for small hydrophobic (non-polar) molecules to be placed into that layer, a process referred to as partitioning.

Partitioning

Hydrophobic organic molecules partition into the hydrophobic region of the CTAB bilayer surrounding a gold nanoparticle.

I have specifically studied the partitioning of the four molecules shown below.

Different naphthol compounds have polar (hydroxy and methoxy) and nonpolar (naphthalene ring) groups.

Different naphthol compounds have polar (hydroxy and methoxy) and nonpolar (naphthalene ring) groups.

The main goal is to quantify how differences in the molecular structure – such as size, polarity, and structure – could affect this partitioning. Hopefully, this will provide insight into how different drug molecules can be utilized to treat tumors and diseases in the medical field.

 

My name is Laura Lee, and I am a rising senior Chemistry major. This is my first summer doing research in Dr. Thompson’s Lab. My project investigates environmental toxicity. The nanoparticle industry is continually growing and has implemented its technology to enhance products people use every day. This is especially true with athletic clothing: silver nanoparticles can now be found in some athletic t-shirts and socks because of their unique antibacterial and odor neutralizing properties. While this can be seen as beneficial to the wearer and those around them, many researchers are looking ahead to the potential consequences of this technology. Think about it: This athletic clothing will still end up in the laundry even if the silver is helping to control the odor. Some amount of the particles will be removed from the clothing after every wash cycle and make their way to a waste water treatment facility. Unfortunately, these facilities are not equipped to filter out such tiny particles (remember: about 600 nanoparticles could fit along the diameter of a human hair). Thus, nanoparticles are washed away into nearby rivers, streams, and other water systems. What happens when these nanoparticles enter the environment and encounter plants, animals, and other chemicals? This is a question Dr. Thompson and many other researchers are trying to answer.

Nanoparticles enter the environment in many different ways from many different sources.

Nanoparticles enter the environment in many different ways from many different sources.

My research this summer is a part of Dr. Thompson’s on-going Tadpole Project. Last year, he studied developmental changes in tadpoles exposed to gold nanoparticles and preliminary research on tadpole uptake of gold nanoparticles. This year, we are solely focusing on quantifying gold uptake. Professor Fong in the Biology department raised two types of tadpoles, bullfrogs and woodfrogs, in two variable tanks – a gold nanoparticle solution and CTAB solution (the coating around the particles) – and a control tank. After the 25-day exposure period, the tadpoles were graciously sacrificed and preserved until I was able to process them. The bulk of my time in the lab has been spent processing the tadpoles and working with Professor Andresen in the Physics department to run samples using ICP-OES. This machine is able to detect and quantify the amount of specific metals – in my case gold – in a given solution. We will use the data to see how much gold is being taken up by the two types of tadpoles and if we can relate it to the size of the species or other characteristics of the tadpole.

 

Bullfrog tadpoles are just one example of creatures that are susceptible to nanoparticles in the environment.

Bullfrog tadpoles are just one example of creatures that are susceptible to nanoparticles in the environment.

My name is Michael Counihan, and I am a rising junior Chemistry and Music double major at Gettysburg College. I am interested in graduate school and doing research in the field of analytical chemistry. This summer is my first research experience, and I am splitting my time between the Frey and Thompson labs investigating lipid monolayers and nanoparticle surface-enhanced Raman spectroscopy, respectively.

Raman Setup

Raman Setup - Cuvette

Our Raman setup: a 785 nm (NIR) laser, Raman spectrometer, computer, and state-of-the-art dark box and cuvette holder.

 

Raman spectroscopy is a unique type of spectroscopy that utilizes inelastic (Raman) light scattering to give information about bonds in molecules. However, Raman signals are very weak since only about 1 in 1,000,000 electrons experience this inelastic scattering (the wavelength of light exciting the electron is different from the wavelength the electron emits as it jumps back down to a different lower energy state). To enhance this Raman scattering up to a million times, molecules can be brought close (within 5 nm) to the surface of certain substrates (in this case, gold nanoparticles); this is known as surface-enhanced Raman spectroscopy (SERS).

Raman signal intensities increase dramatically and a new Au-Br band appears when nanoparticles are introduced to a CTAB solution.

Raman signal intensities increase dramatically and a new Au-Br band appears when nanoparticles are introduced to a CTAB solution.

In Dr. Thompson’s lab, I am synthesizing gold nanorods of different lengths and concentrations to see which particles will give us the best signal enhancement. These particles are surrounded by a CTAB bilayer which keeps them stable. However, the bilayer will begin to collapse when the concentration of CTAB in the solution is too low (around 2-5 mM; aggregation occurs at less than 1 mM). When the bilayer collapses, it is brought closer to the surface of the particle, and signals from the aliphatic tails of the CTAB experience enhancement. My goal is to investigate how adding other molecules into the bilayer (right now, dihydroxynaphthalenes) affects the SERS signals of the CTAB as it collapses and use this data to gain information about this surfactant bilayer.

Bilayer stability is dependent on the concentration of CTAB in the solution.

Bilayer stability is dependent on the concentration of CTAB in the solution.

 

Life in the Lab applied Outside the Lab

               While working in a research lab over the summer is obviously focused on learning about the research, there are many other things that happen when a summer is spent working in a scientific community. We have learned a great deal about communication skills and how to present my research to a broad range of audiences. In addition, the faculty we work with act largely as mentors, providing us with insight on graduate schools and our future endeavors, and bake us cookies once a week to help us make it through the struggles of research. The connections and skills gained this summer will prove useful as we continue on in our studies and in our lives.

Powell Lab: Studying innate immunity in c. elegans

Hello all!

My name is Leah Grandi, and I am the only member of the Powell lab staying this summer. My usual labmates, Joe Robinson and Jimmy Nguyen, are off working at Pitt and Johns Hopkins (aka. places that probably have more windows than our lab here). As for myself, I remain in McCreary 208, with the dull hum of the incubators and freezers as my constant companions.

Figure 1. Our lab family! From left to right, we have myself, Dr. Powell (also known as Glorious Leader or J. Pow), Joe and Jimmy.

Figure 1. Our lab family! From left to right, we have myself, Dr. Powell (also known as Glorious Leader or J. Pow), Joe and Jimmy.

We are molecular geneticists who choose to research with worms. Now, these aren’t the earthworms that you probably thought of initially. These worms are microscopic nematodes known as Caenorhabditis elegans (C. elegans for short). And we think they are pretty cute.

Figure 2. A normal agar plate of C. elegans eating some delicious E. coli. Bon appetit! Image from: http://molecular-ethology.biochem.s.u-tokyo.ac.jp/g1/wt%20on%20plate.jpg

Figure 2. A normal agar plate of C. elegans eating some delicious E. coli. Bon appetit!
Image from: http://molecular-ethology.biochem.s.u-tokyo.ac.jp/g1/wt%20on%20plate.jpg

Our lab uses C. elegans to research the immune system. We are primarily interested in the innate immune system, the part of your body’s defense mechanism responsible for fever and inflammation. One of the most interesting things about the innate immune system to us is the conservation of this system in all animals. That means that this adorable microscopic worm and we complex humans have similar ways of responding to infections. And we think that’s pretty cool.

To test the immune system, we infect our worms with Pseudomonas aeruginosa, a particularly nasty bacteria that is a common cause of hospital acquired infections. Our protein of interest is called FSHR-1, and it has been shown many times to be involved in innate immunity. Worms that do not produce the protein FSHR-1 are immunocompromised compared to worms that do produce FSHR-1.

Another lab that we collaborate with identified a particular toxin that P. aeruginosa secretes called Exotoxin A. This lab also identified two genes that were induced in the worm upon infection with normal bacteria (Escherichia coli, C. elegans’ food) that have been genetically engineered to secrete Exotoxin A. This induction was observed to occur in an FSHR-1 dependent manner. Naturally, we are interested in these genes and want to learn more about them. We have a pretty cool technique to do just that.

One thing that every worm scientist loves to do is make our worms glow. To do this, we fuse the 3’ and 5’ DNA regulatory regions of these genes to the gene for Green Fluorescent Protein (GFP). When our DNA construct is injected into a worm, the worms’ cells will transcribe and translate GFP every time the cell transcribes and translates the gene of interest. We can see the GFP by using a very fancy and expensive microscope (also unfortunately located in a room without windows). The worms that we create are a very helpful genetic tool for visualizing the expression of the gene of interest.

Figure 3. An excerpt from my Celebration 2014 poster, outlining the genetic process of creating a reporter construct.

Figure 3. An excerpt from my Celebration 2014 poster, outlining the genetic process of creating a reporter construct.

Last semester, I worked on creating the DNA constructs for two genes of interest. Dr. Powell managed to inject one of these constructs into a worm. When we infected these worms with P. aeruginosa, we saw some small green spots in the worms’ tail that weren’t there when the worm was eating E. coli. This was very exciting for us, because it means that our reporter works!

However, when the DNA construct is first injected it remains outside of the genome. This means it’s just floating around in the nucleus, and isn’t always transmitted to the next generation because it’s not integrated into any of the worms’ chromosomes. My current project has been to integrate the DNA construct into the worms’ genomes. To do this, I expose the worms to radiation and hope that the DNA gets knocked into one of the chromosomes of that worm’s offspring. So far, I haven’t found an integrated worm yet, but the search is still ongoing.

That’s all for my project! Also while in the lab this summer, I have been doing some other projects, such as a genetic cross for Jimmy and more reporter inductions for Joe.

Please, if you have time and would like to see some cool worms, stop by McCreary 208. I welcome any and all company.

Sincerely,

An extrovert who is working all alone this summer