Table Of ContentSUMMER INSTITUTE FOR LIFE SCIENCES
SIXTH ANNUAL UNDERGRADUATE SYMPOSIUM
ABSTRACTS
August 8, 2016
Presentation Time Noted at End of Abstract
All Talks Will Be In Warren 151
Underlined Author is Presenter
Identification of Possible Erythritol Metabolizing Enzymes
Faviolla A. Báez-Cruz1,2, Martha S. Field2, Patricia A. Cassano2, and Patrick J. Stover2
Stover Lab
Molecular Biology & Genetics Research Experience for Undergraduates
1Department of Chemistry, University of Puerto Rico- Río Piedras Campus, San Juan, Puerto Rico
2Division of Nutritional Sciences, Cornell University, Ithaca, New York
The weight-gain of young adults entering college is more commonly known as the “freshman 15.” In 2011, a group of Cornell
University students residing on campus were assessed upon arrival and then nine months later to compare their health
status. Both whole blood and several anthropometric measures were assessed at both timepoints. Samples were pooled
according to gain weight, HbA1c, and central adiposity. Also, metabolomics analysis was conducted allowing the
identification of metabolites that may be markers of adiposity gain. Among these metabolites, erythritol, was found elevated
in students who gained more weight. Erythritol is an alcohol sugar, observed in serum at concentrations high enough to
suggest it may not have been acquired through diet alone, indicating that the body produces it. Subsequent experiments
using isotopically labeled glucose revealed that humans convert glucose to erythritol. Approximately 5-10% of eyrithritol is
excreted, but the fate of the remaining erythritol and an enzyme responsible for metabolizing it remain unknown. Yeast
homologues of NADPH-dependent proteins AK1A1 and AK1B1, from the aldehyde and aldose reductase families, are known
to metabolize erythritol. The proposed reactions reduce an aldehyde or an aldose, respectively, to an alcohol form, using
NADPH; although the reverse reaction could occur with NADP+. We cloned and transformed each cDNA into both bacterial
and mammalian cells. Preliminary experiments indicate that recombinant AK1A1 and AK1B1 can be purified and over-
expressed in bacterial cells. AK1A1 did not metabolize erythritol, though the enzyme was active with known substrate
glyceraldehyde. Studies with recombinant AK1B1 are ongoing to determine if it is a mammalian erythritol-metabolizing
enzyme. 1:54-2:06 PM
Evaluating the interaction between a key germline stem cell regulating gene and Wolbachia
Kristen Rose Baxter, Jaclyn Bubnell, Charles Aquadro
Aquadro Lab
Department of Molecular Biology and Genetics
Germline stem cells (GSCs) are cells that either renew as stem cells or differentiate into egg or sperm. In Drosophila,
differentiation is controlled by the GSC regulatory gene bag of marbles (“bam.”) bam has been shown to be evolving rapidly
due to positive selection in D. melanogaster and D. simulans. This is interesting because bam plays a critical role in
reproduction. One possible force driving this adaptive evolution is the presence of Wolbachia pipentis, an endosymbiotic
bacteria that lives in Drosophila germline cells and that can manipulate host reproduction.
Past research in our lab has shown that the presence of Wolbachia can rescue fertility in D. melanogaster females with a
mutant copy of bam that causes tumorous ovaries. I am conducting assays with bam hypomorphs and bam knockdowns to
assess Wolbachia’s effect on female fertility throughout the fruit fly’s lifespan. 3:54-4:06 PM
Making “scents” of the olfactory environment
Marlene Berke1, Thomas Cleland2, David Field1.
1The Field Lab
Department of Psychology, Cornell University, Ithaca, NY. 2Computational Physiology Laboratory (CPL), Department of
Neurobiology and Behavior/Department of Psychology, Cornell University, Ithaca, NY.
A fundamental question in neuroscience is how the brain encodes and stores sensory information in order to remember and
later recognize stimuli. Sparse coding presents a solution. Sparse coding is an object-recognition algorithm that sensory
systems may use. In order for sparse coding to apply, the stimulus environment must have a particular property: sparseness.
Our lab simulates olfactory worlds and shows that they are sparse. When applied to suitably sparse data, such as those
generated by our simulations, the sparse coding algorithm results in a population of detectors that learn (without
supervision) to respond selectively to specific stimulus sources. Taken to its extreme, sparse coding could result in an apple
detector neuron, for instance. Substantial evidence for sparse coding has been found in the visual system. We believe that
sparse coding is also used by the olfactory system.
Sparse coding requires that the sensory input data have a sparse structure. In other words, a state space of possible inputs is
not uniformly filled: in olfaction, this means that some odors are often encountered, while others are never encountered. We
used a series of simulations in order to show how sparsely structured data can arise from a very simple olfactory world.
The signature of sparse coding is a layer of neurons showing population and lifetime sparseness — that is, only a few neurons
out of the population are active at any given time, and each neuron is rarely active. The piriform cortex, like the primary
visual cortex in visual systems, shows this kind of sparseness. However, as in visual systems, the first layer of receptors —
Olfactory Sensory Neurons, or OSNs — does not show any apparent sparseness. We hypothesize that, because of the
sparseness in the environment and the way animals explore it, the signal represented by the OSNs must be sparse, even if the
neurons themselves do not show sparse activity when recorded over short periods of time. Instead, the sparseness of the
signal is independent of the sparseness of the representational medium. We hypothesize that evidence of the sparse signal
will be found in the activation patterns of the population of OSNs over time — because some odors are encountered
frequently, and others not at all, some patterns of receptor activity occur frequently, and others not at all. This results in a
sparseness of the signal in receptor space, making the data tractable for sparse coding. 3:30-3:42 PM
Engineering glycosylation of proteins in Escherichia coli
Aravind Natarajan1, Marielisa Cabrera1, Matthew DeLisa2
DeLisa Lab
1Department of Microbiology, 2School of Chemical and Biomolecular Engineering
In the face of an ever-growing assault of pathogens and diseases, there is a pressing need for the rapid development of novel
and inexpensive therapeutic solutions. Proteins are widely considered workhorses, leveraged as vaccines, hormones and
drugs in the maintenance of human health. Among these, recent advancements have proven that proteins with covalently
bound glycans, glycoproteins, in particular can pave way to the cure of a host of diseases such as cancers and genetic
disorders associated with the nervous system. There are two major types of glycoproteins - glycans bound to the amide
group of an asparagine amino acid (N-linked) and those bound to the hydroxyl group of a serine or threonine amino acid (O-
linked).
Current in vitro strategies to produce glycoproteins including chemical synthesis and chemo-enzymatic methods are
expensive and not easily scalable. Furthermore, in vivo synthesis methods in plants, yeast cells and mammalian cell lines
produce uncontrollable heterogeneity in glycans. The inability to produce glycoproteins with homogeneous glycans is an
impediment to the production of high affinity therapeutic antibodies that could be used to combat cancer and genetic
disorders of glycosylation.
Our research focuses on engineering glycosylation pathways in Escherichia coli transforming these bacteria into a “factory”
that can produce glycans and glycoconjugates by design. We recently engineered E. coli to produce N-glycoproteins
containing trimannosyl chitobiose glycans generally found in the human body, and hitherto unfound in bacteria. I am now
working to constitute an O-linked glycosylation pathway. Taken together, we aim to create a platform in E. coli for the precise
and scalable production of therapeutically relevant glycoproteins. 11:30-11:42 AM
Elucidating the Transcriptome: Grapevine Red Blotch-associated Virus
Antonio Cerullo, Dr. Keith Perry, Dr. Jeremy Thompson, Jose Asencio Vargas
Perry Laboratory
Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science
Microbial Friends and Foes REU Program
Grapevine Red Blotch Disease was first observed in 2008 and the causal agent named Grapevine Red Blotch-associated Virus
(GRBaV). This virus has been detected in numerous cultivars across the United States and is found to alter fruit chemistry,
flavor, and yield. Currently, very little is known about this virus’s replication. My primary objective was to evaluate GRBaV
transcription by northern blot analysis. To address this we did 1. large-scale nucleic acid extractions (>mg quantities) of grape
leaf tissue, 2. RNA hybridization utilizing chemiluminescence, and 3. 5’ termini determination of transcripts. Nucleic acid
extraction followed by DNase treatment results in the isolation of mRNA molecules present in grapevine tissue. Following the
agarose gel electrophoresis of RNA and transfer to a nylon membrane, hybridization using chemiluminescent methods
identifies the number and size of RNAs synthesized from each targeted open reading frame. Specifically designed
hybridization probes detected viral RNA transcripts. Rapid Amplification of cDNA Ends (RACE) PCR of reverse-transcribed
cDNA can be used to identify the 5’-ends of transcripts. Amplicons were cloned into Escherichia coli using the pGEM®-T
vector and sequenced. Continuation of this study will further understanding of Geminivirus transcription and genetics.
10:06-10:18 AM
Investigating the role of the regulatory elements in the upstream region of PSPTO_5255 in Pseudomonas syringae pv.
tomato strain DC3000
Alexa Cohn, Melanie J. Filiatrault
Filiatrault Lab
Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science
Microbial Friends and Foes REU Program
Pseudomonas syringae pv. tomato strain DC3000 (Pto) is a pathogen of tomatoes, causing tomato speck, a disease
characterized by brown-black leaf spots surrounded by chlorotic margins, sunken specks on fruit, stunting, and overall yield
loss. During infection, the bacteria encounter high levels of Ca2+ in the plant apoplast. A two-component system (TCS)
consisting of a histidine kinase (HK) sensor and a DNA-binding response regulator (RR) in Pto consisting of cvsS (HK) and cvsR
(RR) is induced by high levels of Ca2+ and plays a role in virulence. ∆cvsS and ∆cvsR strains show decreased virulence,
increased biofilm production, decreased swarming, and the formation of Ca2+ precipitate on the colony surface.
A gene found to be in the cvsR regulon, PSPTO_5255 (5255), encodes a carbonic anhydrase. A Δ5255 mutation results in
phenotypes similar to those of the ∆cvsS and ∆cvsR strains. 5’ RACE, a technique used to amplify the 5’ ends of cDNA,
revealed two shorter transcripts (112 and 380 bases) in the 5’ region of the 5255 transcript. It is unclear whether the coding
region of 5255 or one the 5’ small transcripts is responsible for the phenotypes seen in the mutant strain. Additional
bioinformatics analysis identified a promoter upstream from the 5255 coding region that has a predicted binding site for the
sigma factor RpoF.
In order to ascertain if RpoF is the sigma factor regulating the transcription of PSPTO_5255, wild type and ∆rpoF strains were
transformed with a plasmid containing a luciferase reporter gene fused to the PSPTO_5255 promoter and then incubated in
conditions to activate the cvsS/cvsR TCS. The ∆rpoF strain showed a lower level of luminescence, suggesting that RpoF is
involved in regulating transcription of PSPTO_5255. Research is ongoing to characterize the phenotypes of strains with
deletions in the coding region of 5255, and sequences corresponding to the two shorter transcripts. The deletion mutants will
be tested for the phenotypes related to calcium precipitation, biofilm formation, and motility. Finally, a translational fusion
between a FLAG peptide tag and a region of interest (either of the shorter transcripts, or the PSPTO_5255 gene) will be
created to test if the truncated transcripts form translated products.
Overall, this project will help to understand some of the regulatory mechanisms used by Pto during infection, as well as to
discern the role PSPTO_5255 plays in the phenotypes related to virulence. 9:06-9:18 AM
Differential Expression Analysis of mRNA-seq and rRNA depleted RNA-seq of C. elegans SET-9 and SET-26
Methyltransferases Mutants
Marcio Jose de Carvalho1,2, Wenke Wang2, Siu Sylvia Lee2
Lee Lab
Molecular Biology & Genetics Research Experience for Undergraduates
Federal University of ABC, Santo Andre, SP, Brazil1, Department of Molecular Biology and Genetics, Cornell University, Ithaca,
NY2
The methylation of histones is a well known component in the regulation of transcription in eukaryotic cells, and this process
is carried out by a complex of enzymes, but mainly, by proteins known as methyltransferases. By understanding how these
enzymes act, we will gain new insights into how chromatin and epigenetic regulation can affect important life history traits,
such as reproduction and longevity. Using the powerful genetic model C. elegans, we have identified the methyltransferases
SET-9 and SET-26 to have critical roles in fertility and lifespan.
Mutations were introduced in both set-9 and set-26 genes to create three different loss of function strains of worms, set-9(-),
set-26(-), set-9/set-26(-/-), that then had their lifespan and fertility phenotypes quantified in comparison to the normal(wild
type, wt) worms. Because methyltransferases are expected to regulate chromatin structure and ultimately gene expression,
it is critical to analyze the global gene expression profiles in these mutant strains, to identify the genes that show significant
expression change. The RNA-seq approach is a way to quantify gene expression levels by using next generation sequencing
technique. There are three main methods for RNA-seq that differ in how they remove the ribosomal RNAs before sequencing
the other classes of RNAs. From these, we chose mRNA-seq, which is based on poly-A enrichment for processed, translation-
ready mRNAs, and RiboMinus RNA-seq, which works by tagging and removing the ribosomal RNAs. The latter method has the
added value of quantifying RNAs that do not have polyA tails but nevertheless have key biological roles.
The goal of my project was use statistical analysis pipelines to determine the differentially expressed genes identified by the
two different methods, and then perform further downstream bioinformatics analysis to reveal the function groups and
pathways that those genes belong to. The rRNA-depletion method yielded more data, as expected, revealing some biological
functions that might be affected by SET-9 and SET-26. A possible direction now is to further research how these biological
functions are connected to the observed phenotypes of longevity and sterility. 1:30-1:42 PM
How Fat Accumulation Relates Fertilization of C. elegans
Carlos G. Declet Nieves1, Amaresh Chaturbedi2, Siu Sylvia Lee2 University of Puerto Rico Rio Piedras Campus, San Juan, PR1,
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY2
Lee Lab
Molecular Biology & Genetics Research Experience for Undergraduates
Department of Molecular Biology and Genetics
The ability of organisms to control and regulate fat storage is a key for their survival. It is known that fat accumulation and
obesity are associated with serious health conditions in some organisms, because it may contribute to insulin resistance,
cardiovascular conditions, and some types of cancer. In the research carried out, the main purpose was to study the
relationship between sterility and fat accumulation in a microscopic nematode, C. elegans, which has shown to be a powerful
model for the study of genomics relevant to mammals. Reproduction is an energy-intensive process and fat is one of the
sources for energy. We hypothesize that sterility would cause hyper accumulation of fat, because the inability to breed
offspring will lessen energy expenditure. To test this, (i) we performed RNA inference (RNAi) for the genes known to cause
sterility when knocked down and (ii) checked the levels of fat by staining the fixed worms with oil red O, which stains lipids.
This study is important because it will reveal the relationship between fat metabolism and sterility, if any, and will help us
understand how fat metabolism might affect fertilization, health, and lifespan. 1:42-1:54 PM
Metabolic Fate of DHA in Human cells: Direct Retroconversion of 22:6n-3 to 20:5n-3 Dominates Over Elongation to 24:6n-3.
Hui Gyu Park, Peter Lawrence, Matthew Engel, Kumar Kothapalli, J. Thomas Brenna.
Brenna Lab
Division of Nutritional Sciences
Docosahexaenoic acid (22:6n-3) supplementation in humans causes 20:5n-3 to rise in plasma but not in neural tissue where
22:6n-3 is the major omega-3 in phospholipids. We determined whether neuronal cells (Y79 and SK-N-SH) handle 22:6n-3
differently from non-neuronal cells (MCF7 and HepG2). 13C-22:6n-3 was primarily esterified into cell lipids. Retroconversion
was 5-6 fold greater in non-neural compared to neural cells, and retroconversion predominated over elongation by 2-5 fold.
Putative intermediates 13C-22:5n-3 nor 13C-24:5n-3 were not detected. MCF7 cells abundantly expressed mitochondrial
CPT1A, ECI1 and DECR1, whereas peroxisomal ACOX1 was abundant in HepG2 cells, suggesting the initial site of 22:6n-3
oxidation depends on cell type. Non-neural cells more actively metabolize 22:6n-3 to 20:5n-3 via channeled retroconversion,
while neural cells retain 22:6n-3. 4:06-4:18 PM
Elucidating the functions of transcribed and non-transcribed enhancers
Alaa Farghli1,2, Nathaniel Tippens1, John Lis1
Lis Lab
Molecular Biology & Genetics Research Experience for Undergraduates
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853
2Department of Chemistry, SUNY Potsdam, Potsdam, New York, 13676
Promoters and enhancers are regions of the genome that regulate gene expression and thus encode cellular programs.
Hence, promoters and enhancers are critical for maintaining cell homeostasis. Initial views dictated that the promoter is a
region of DNA where RNA transcription begins, and enhancers are regions that can upregulate the expression of a gene.
Despite this extensive research, more recent studies have shown the existence of active enhancers that are transcribed to
produce eRNA (enhancer RNAs), similarly to promoters. These revelations have challenged the established conceptions of
enhancers, and require additional experiments to understand the role of eRNAs. We set out to study whether non-
transcribed regions predicted to be enhancers can promote upregulation of genes, or whether all functional enhancers are
transcribed in a similar fashion to promoters.
To tackle these questions, we use the heat shock response (HSR) as a model system to study the activity of RNA Polymerase II
(RNAP II). The heat shock response combats the adverse effects of heat (such as protein misfolding). Established consensus
motifs (NGAAN) are recognized by transcription factors (TFs), that aid in the transcription of RNAP II in the event of heat
stress environment. This research aims to utilize the genomic editing tool, CRISPR/Cas9, to mutate the consensus motifs in
the enhancers of KLHL26 (non-transcribed) and HSPB1 (transcribed). As a comparison, we will mutate the HSE in the
promoter of HSPB1. These mutants will be compared with the wild-type prior to heat shock to rule out any off-target effects.
Future research will analyze these mutations by ChIP-seq, PRO-seq, and Hi-C, to better understand the differences between
enhancers and promoters. 2:06-2:18 PM
Acyrthosiphon pisum feeding decisions and the role of Pseudomonas syringae
Rachel Fay and Tory Hendry
Hendry Lab
Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science
Microbial Friends and Foes REU Program
This study was undertaken to determine the feeding decisions of aphids in the presence of Pseudomonas syringae, since
epiphytic bacteria have the potential to be lethal to aphids. Some bacterial plant pathogens, particularly the bacterium P.
syringae, form epiphytic populations on the leaf surfaces, which are then ingested by the aphids and multiply until they reach
a lethal dose within the aphid. In this study we sought to determine how aphid feeding behavior was influenced by the
presence of different P. syringae strains in both in vito and in vivo experiments. The two strains utilized in this study, P.
syringae pv. syringae B728a (Psy B728a) and pv. tomato DC2000 (Pto DC3000), are known to differ in virulence, both of which
are virulent to pea aphids. During in vitro experiments, aphids were given no choice of diet (Control, B728a, DC300 each
separately), three choices (control, B728a, DC300), and two choices (B728a, DC3000) in artificial feeding sachets. Throughout
in vivo experiments, surface inoculation was performed on fava bean plants using B728a and control buffer. The number of
aphids feeding on the more virulent strain, B728a, was found to be significantly lower than those recorded feeding on DC300,
a less virulent strain, and the control throughout all in vitro experiments. This study shows that the presence of P. syringae
and the virulence of different P. syringae strains, can influence aphid feeding behavior. Pea aphids feed on a wide range of
plants, such as soy bean, broad bean, pea and snap beans, and in general aphid species can be serious agricultural pests. This
work shows that by killing aphids or deterring aphid feeding, P. syringae may have important consequences for aphid
populations and therefore their host plants. 9:18-9:30 AM
Building tools for efficient tissue-specific gene deletion in Drosophila
Rushaniya Fazliyeva, Tireniolu Onabajo, Yue Qiu, Chun Han
Han Lab
Weill Institute for Cell and Molecular Biology, Department of Molecular Biology and Genetics, Cornell University
Understanding how genes control development of specific tissues is an important task of developmental biology. However, it
is challenging to remove gene functions in a tissue-specific manner. Currently there are two popular methods in Drosophila
melanogaster (fruit fly) to achieve tissue-specific loss of function (LOF): RNA interference (RNAi) and Mosaic Analysis with a
Repressible Cell Marker (MARCM). RNAi suppresses gene expression by knocking down the mRNA. This method is tissue
specific but often suffers from incomplete LOF. While MARCM usually allows for more complete LOF by creating mutant cells
in otherwise wild type animals, its complicated genetic system makes it difficult to study cell-cell interactions. In this study we
aim to build a new method based on CRISPR/Cas9 to achieve efficient tissue-specific LOF of any gene. This strategy is
composed of a tissue specific Cas9 and a pair of ubiquitously-expressed guide RNAs (gRNAs) targeting the genomic DNA of
the interested gene. Cas9-mediated cutting of the genomic DNA can lead to deletion of the gene locus in a tissue-specific
manner. We have generated a number of transgenic lines that express Cas9 in various tissues. To identify the most efficient
Cas9 strains, we developed two assays to evaluate the tissue-specificity of Cas9s and completion of gene deletion based on
green fluorescence protein (GFP) reporters. For each tissue specific Cas9, we identified the best candidate. These Cas9s will
be combined with neuronal type-specific markers to build tools for efficient and specific gene LOF in Drosophila. These tools
will provide us great power in revealing how endogenous genes control neuronal morphogenesis. 3:42-3:54 PM
Cobalt(III) Complexes as Therapeutic Agents in Hypoxic Environments
Hendryck Gellineau, Paden King
Wilson Group
Department of Chemistry and Chemical Biology
Tumor hypoxia is oxygen deficiency within solid tumors. This deficiency occurs when rapidly-dividing tumor cells outpace the
supply of oxygenated blood. Hypoxia confers resistance to numerous methods of radiotherapy and chemotherapy, and also
plays a significant role in metastasis. Therapeutic and diagnostic agents which are active in hypoxia are now being developed
to tackle the problem. These agents target tumor biomarkers such as low pH, reduced pO2, and leaky vasculature. We
approached the problem by synthesizing Co(III) complexes as cytotoxic prodrugs. Our strategy was to take advantage of the
intracellular reduction of Co(III) to Co(II) which causes the breakdown of the prodrug complex, yielding the ligands as drugs in
their free form. To this aim, we synthesized Co(III) complexes using bis(thiosemicarbazones) as equatorial ligands and various
cytotoxic nitrogen donating sigma donors as axial ligands specifically Ammonia, Imidazole, benzylamine, and
nitrobenzylamine. Each bis(thiosemicarbazone) and axial ligand used in a complex modifies the reduction potential of the
metal center, thus increasing the metal’s electron affinity and affecting complex stability. Further electrochemical and
stability studies will be performed to determine reduction potentials and stability constants for each complex. Co(III)
complexes will also be tested for cytotoxicity in different cell lines under both hypoxic and normoxic conditions. The
cytotoxicity of each complex will be measured by MTT assay, a colorimetric assay that assesses cellular metabolic activity
through the reduction of a tetrazolium dye, producing a purple color indicative of living cells. Synthesis of a successfully
hypoxia-active cytotoxic prodrug would open new avenues of therapy in solid tumors. 11:54-12:06 PM
The Role of β1-Integrin in Bladder Smooth Muscle Function
Sarah Hanif1, 2,3, Warren Hill PhD2, 3, Bryce MacIver PhD2, 3, Mark Zeidel MD2, 3
Cornell University1, BIDMC2, Harvard Summer Research Program in Kidney Medicine, Harvard Medical School3, Boston, MA
The mechanism by which the bladder senses wall stretch is not well understood. Integrins are transmembrane proteins that
physically link intracellular actin to the extracellular matrix (ECM), and are heavily involved in cellular sensing of force.
Expression of integrins and ECM constituents has been shown to change in animal models of bladder overdistension and
diabetes, and blockade of integrin function inhibits stretch-stimulated DNA synthesis. This suggests that regulation of smooth
muscle (SM) proliferation may depend upon mechanical cues from the ECM.
To determine the role of integrins in normal bladder function, we began characterizing a genetic mouse model that enables
the tamoxifen-inducible knockout of smooth muscle β1-integrin using Cre-LoxP targeted gene deletion.
We i.p. injected six C57Bl6/J mice and six B6.FVB-Tg(MyhII-cre/ERT2)1Soff/J/b1-intfl/fl (MyhII-Cre) mice with 2 mg tamoxifen
for 6 days. We tested for changes in bladder phenotype by performing void spotting assays on filter paper for 14 days.
Bladders were then harvested for analysis.
Our results demonstrated a 70% reduction in integrin protein expression (p<0.01) by Western blot after 14-days and RT-PCR
confirmed deletion of exon 3 from β1-integrin mRNA. Although H&E staining showed potential hypertrophy of the SM in
knockout mice, void spotting assays did not show a change in urination pattern.
We anticipate that a longer induction period following tamoxifen may result in greater reduction of integrin expression and
allow determination of a phenotypic change. This model shows promise for in-depth studies aimed at understanding the role
of integrins in bladder smooth muscle pathology. 4:18-4:30 PM
Genetic approach for identification of genes that influence AlgU-dependent down-regulation of fliC expression in
Pseudomonas syringae pv tomato DC3000
Elena Holley, Eric Markel and Bryan Swingle
Swingle Lab
Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science
Microbial Friends and Foes REU Program
Pseudomonas syringae are common environmental bacteria that are host-specific plant pathogens. The DC3000 strain of P.
syringae is a tomato-specific pathogen and has been fully sequenced, allowing for research based on genomic analysis.
Motility of the bacteria through flagella is encoded by the fliC gene, which is suppressed by the AlgU sigma factor. However,
previous research in our lab indicates AlgU is unlikely to directly control fliC expression. Therefore, the purpose of this
research was to identify other genes necessary for AlgU-dependent down-regulation of fliC expression. We used a genetic
approach to identify genes that, when disrupted, prevent AlgU from being able to down-regulate fliC expression. For this
screen, we constructed a plasmid in which the fliC promoter was fused to the gus reporter gene. The function of this plasmid
was confirmed by demonstrating that clones grown on media containing Xgluc developed a blue color in the absence of AlgU
and were not blue when AlgU was expressed. To find genes necessary for AlgU-dependent down-regulation of the fliC
promoter, we used the AlgU overexpression strain containing the PfliC::gus fusion to generate a transposon insertion library.
This was then screened for mutants that were no longer able to repress gus expression and were, therefore, blue on media
containing Xgluc. A total of about 20,000 colonies were screened, of which 858 were blue. The 117 darkest blue clones were
chosen for further analysis. Arbitrary PCR and sequencing were used successfully to determine the location of the transposon
insertion for 93 of the dark blue clones. These results indicated that insertions in 12 different genes altered AlgU-dependent
control of the fliC promoter. Most noteworthy was the gene coding for the sigma factor SigX, as well as its neighboring gene,
oprF. The regulatory connection between oprF and algU is yet unclear, whereas this type of connection between sigX and
algU has previously been shown through ChIP-sequencing. Next steps would include further testing of the regulation of gene
expression among algU, sigX, and fliC. Additionally, it may be useful to test the transformants using a lux reporter vector in
order to quantifiably determine the degree of regulation they hold in the algU-fliC pathway, as well as testing their AlgU-
dependent phenotypes, including motility and alginate production. 9:30-9:42 AM
Genetic engineering of Escherichia coli to combat mastitis in cows
Cornell iGEM Team
Department of Biomedical Engineering
Mastitis is the most common and costly disease in animal agriculture, and causes the U.S. dairy industry to lose $2 billion
annually. An invasion of the teat canal by bacteria results in the inflammation of the mammary gland, which renders the milk
unusable. In many cases, mastitis also contributes to the cattle’s death. Farmers currently combat mastitis through
preventative methods, and treat the inevitable infections with antibiotics. Antibiotics, however, leave traces in milk, and can
have cure rates as low as 15%.
Through a novel synthetic biology approach, Cornell iGEM aims to combat mastitis by genetically engineering Escherichia coli
to produce natural antimicrobial peptides, bacteriocins, against some of the most prevalent and historically significant
pathogens of mastitis. Bacteriocins function like natural antibiotics with distinct advantages. First, whereas most antibiotics
tend to be broad-spectrum in nature, bacteriocins are usually very specific to a few closely related species. Second, although
the usage of antibiotics may have implications in human well-being, bacteriocins are known not to have negative effects on
human health. Finally, where the discovery of new antibiotics is stagnating, bacteriocins are constantly evolving to reflect the
Red Queen hypothesis among bacterial species. These characteristics render bacteriocins a much more attractive treatment
option. We intend to perform simple zone of inhibition and growth curve assays to assess the efficacy of the purified
bacteriocins. After initial testing, we will analyze quantitatively the both the minimum inhibitory concentration and the
stability of the bacteriocins.
To ensure that our project assists current dairy farmers, the Cornell iGEM team has been gathering feedback from local farms
and companies. To supplement the bacteriocin treatment, the Cornell iGEM team is also creating an app for farmers and
developing a modified shell for milking consisting of separate modules. Through all of these components, we hope to
eliminate setbacks that dairy farmers face in providing the world with food. 11:42-11:54 AM
Neural Tracing and Recording in the Dorsal Raphe Nucleus
Nicholas Krupa, Changwoo Seo
Warden Lab
Department of Neurobiology and Behavior
Despite being a widespread and potentially debilitating mental disorder, the mechanics behind depression and its related
behaviors are still not well understood. Serotonin is a neurotransmitter that has long been suspected of being connected to
the disorder, with several of today’s most common antidepressants being Selective Serotonin Reuptake Inhibitors (SSRIs),
which inhibit the reuptake of serotonin by presynaptic neuronal membranes. While this has been shown to provide relief
from depressive symptoms, it is again not well known exactly how this occurs. In order to further understand the mechanics
of depression, and potentially to create more effective treatments, its link to serotonin must be explored.
To this end, our work targets the dorsal raphe nucleus, a major serotonin center in the brain. Through the use of mice as
models of human depression, we seek to determine how serotonergic neurons (and neurons that they connect to) ‘react’ in
different behavioral contexts. In order to identify neurons of interest, viral vectors and tracers are injected into the brains of
living mice. Using different combinations of these tracers, as well as different strains of transgenic mice, we are able to
determine the degree to which the DRN projects to other certain areas, and to which other areas project to the DRN. In order
to record the activity of suspect neurons, specific viral vectors are injected that cause infected neurons to fluoresce when
high concentrations of calcium are detected (such as when the neuron is activated). By also inserting a small optical fiber into
the brain, the frequency of these activations can be recorded while the mouse is free to function as normal; this is a
technique known as fiber photometry.
With these techniques combined, the activity of serotonergic neurons and the circuits they belong to can be closely examined
in varying contexts, including mice that express depressive symptoms and those that do not. Through this level of
examination, it is hoped that the link between serotonergic neurons and the occurrence of depressive symptoms can be
better understood, in order to gain a clearer picture of how the disorder of depression actually functions. 4:30-4:42 PM
Mutant Verification of Quantitative Disease Resistance Genes of Northern Corn Leaf Blight
Micayla Menchel, Tyr Wiesner-Hanks, Dr. Judith Kolkman, Dr. Rebecca Nelson
Nelson Lab
Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science
Microbial Friends and Foes REU Program
Northern Corn Leaf Blight (NCLB) is a foliar disease of maize caused by the fungus Setosphaeria turcica. NCLB can cause
dramatic corn yield loss due to characteristic necrotic lesions that decrease photosynthetic output. While genes conferring
qualitative resistance to NCLB have been widely utilized by breeders, the rapid proliferation of new S. turcica races has
resulted in the breakdown of the effectiveness of these genes. It is important to locate genes conveying quantitative disease
resistance (QDR) as they offer new opportunities for breeders to develop more robust NCLB-resistant maize lines.
Previously conducted genome-wide association (GWA) and nested association mapping (NAM) studies have indicated regions
of the maize genome where single nucleotide polymorphisms (SNPs) are consistently and significantly associated with
differences in disease phenotype. By combining multiple mapping and quantitative trait loci (QTL) linkage studies, potential
target genes impacting NCLB disease phenotype have been identified. In order to verify potential genes and eliminate false
positives, we performed reverse genetic screens using the UniformMu population. Insertions of the Mutator transposon in a
uniform inbred background can knockout specific genes by disrupting regulatory elements or transcription machinery.
We genotyped forty-eight families by PCR that have been screened for NCLB resistance in the field for two years and further
evaluated nine families with insertions in one of seven genes of interest in the greenhouse. Thirty of the forty-eight field
families were successfully genotyped and the impact of known mutant genotypes on area under disease progress curve
(AUDPC) was then analyzed. All nine greenhouse families were successfully genotyped and analyzed in comparison to
incubation period. Five of the thirty-nine families screened showed a significant difference between mutant and non-mutant
genotypes on disease phenotype. All five genes were located surrounding historically significant QTLs on three chromosomes:
An F-box domain protein in bin 1.02, a guanylyl cyclase and uncharacterized protein in bin 6.05, and a calcium- dependent
metal ion binding domain and uncharacterized protein flanking bin 8.06.
These findings suggest that QTLs are composed of clusters of genes that may or may not be directly related. They also suggest
that QDR is multi-mechanistic and genes that play a role in QDR may be spread across the genome. Identification and
verification of specific genes involved in QDR allows for further investigation into the exact roles and processes performed by
these genes in plant defense. 10:18-10:30 AM
Investigating the function of cis-regulatory elements in mammalian 3'UTRs
Mauricio Paramo,1,2 Rene Geissler,1 and Andrew Grimson1
Grimson Lab
Molecular Biology & Genetics Research Experience for Undergraduates
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA; 2Department of Biological
Sciences, Binghamton University, Binghamton, New York 13902, USA
The predominance of post-transcriptional dysfunction in numerous genetic disorders and cancers makes gene regulation at
the level of the RNA a central area of investigation. Post-transcriptional control is driven by cis-acting regulatory sequences
preferentially located within the 3' untranslated region (3'UTR) of mammalian messenger RNA (mRNA). These sequences
recruit trans factors, such as noncoding RNAs (e.g. microRNAs) and RNA binding proteins, that affect mRNA processing,
stability, localization, and translation. Recent comparative genomic studies and high-throughput cell-based screens have
identified hundreds of novel cis-regulatory elements, but the mechanisms through which these elements influence gene
expression remain unknown. Here, we focused on the characterization of a subset of sequence elements that activate gene
expression post-transcriptionally. We selected candidate activating sequence elements that lead to increased reporter levels
but did not significantly increase mRNA levels. To focus on elements most likely to possess biological significance, we
furthermore required that our candidate elements be evolutionarily conserved in mammalian 3'UTRs. We validated the post-
transcriptional activating potential of each candidate element using both transient and integrated reporter constructs in
human cell lines. The results of our work will help elucidate how multiple activating post-transcriptional cis-regulatory
elements influence gene expression, thus expanding the set of known sequence elements that play important roles in
influencing mammalian gene expression. 2:18-2:42 PM
Effect of landscape complexity on ground beetle (Coleoptera: Carabidae) diversity and body size in Central New York
Anthony Polyakov, Ricardo Perez-Alvarez, Katja Poveda
Poveda Lab
Department of Entomology
Agricultural landscape complexity is an important measure of the natural areas surrounding a cultivated field. Complex
landscapes contain a greater abundance of natural areas compared to simple landscapes, which are primarily composed of
cultivated land. These natural areas are thought to provide generalist arthropod predators with additional resources, which in
turn contribute to their diversity and abundance within agricultural landscapes. Ground beetles, which are generalist
predators commonly found in agricultural fields, have been observed to increase in diversity and body size across complex
landscapes. However, most of the prior studies conducted thus far have mainly focused on a few particular species, without a
clear picture of the community dynamic. This study aims to address this gap in knowledge by studying how entire ground
beetle communities are affected by landscape complexity across the Finger Lakes region.
In our system, ground beetles were sampled in twelve cabbage fields using pitfall traps in the months of June through August
of 2014. Then the ground beetles were separated from other arthropods and sorted into plastic alcohol vials. Using different
sized insects pins, these ground beetles were pinned and organized in different boxes according to the farm they were
collected from. After being pinned, the width of the pronotum, length of the elytra, and length of the hind femur were
measured on the ground beetles utilizing a built-in ruler from a microscope. While these measurements were conducted, a
taxonomical key to ground beetles of northeastern North America (Coleoptera: Carabidae) was used to aid in identifying and
sorting out the different ground beetles to species level. Our preliminary results showed that there are at-least 50 species of
ground beetles when combining all twelve cabbage fields. The most abundant species observed was Bembidion
quadrimaculaum, with 427 out of 1676 total individuals. We also noticed that the total abundance was negatively correlated
with landscape complexity, but some species did not adhere strictly to this pattern. With over a thousand carabid beetles
already measured, this project is still a work in progress, and a comparative analysis of the body sizes of the different species
will be conducted in the near future. Once all the specimens are fully identified to species level, and all the relevant
specimens measured in their body size, then a community level analysis will be carried out. 3:06-3:18 PM
The influence of early social environment on juvenile egression in Microtus ochrogaster
Jeanne Powell, George Prounis, Alexander Ophir
Ophir Lab
Department of Psychology
Prairie voles (Microtus ochrogaster), a rodent species found abundantly in the Midwest, are one of the few socially
monogamous species of mammals. Usually, prairie voles display robust bi-parental care. However, natural variation in this
family structure establishes the prairie vole as a popular model organism for studying the influences of early-life social
environments on the development of offspring. The aim of our research is to explore which factors impact a juvenile’s
decision to leave its natal nest. Our current experimental paradigm is allowing us to explore the relationship between
juvenile space-usage and their parental environment (i.e. being reared by a single-mother or bi-parentally). While we are still
in the process of acquiring and processing data, we have already seen interesting and marked variation in the space use of
the pups. Based on previous field research, we predict that pups reared by a single mother will be more exploratory than
those reared bi-parentally and that parental and pup behavior will be highly correlated. 3:18-3:30 PM
Description:later recognize stimuli. Sparse coding presents a solution. Sparse . Putative intermediates 13C-22:5n-3 nor 13C-24:5n-3 were not detected. MCF7 cells abundantly .. sized insects pins, these ground beetles were pinned and organized in different boxes according to the farm they were collected from.
