6V��Ӱ��

Many students in the Molecular Biology program undertake focused research during the summer through 6V��Ӱ��'s Summer Undergraduate Research Program. Below are recent completed projects by our students.

2019

The Impact of β-integrin, e-cadherin, and β-catenin Knockdown on Drosophila Larval Hemocytes

Brenda Wong ’21; Advisor: Cris Cheney

Preliminary observations from the Cheney Lab indicated that the knockdown of rab5 reduced the number of hemocytes in the cuticle pockets, in addition to an overabundance of circulating hemocytes. One hypothesis may be that the rab5 knockdown, 30518, blocks the cell adhesion molecules. The goal of this project is to determine whether knocking down the cell adhesion molecules-β-integrin, e-cadherin, and β-catenin-have similar effects to the rab5 knockdown. This was done by counting the number of hemocytes per larvae and imaging the hemocytes of the larvae.

Domeless and Eater Knockdowns on Hematopoiesis

Jose Carranza Celis ’22; Advisor: Cris Cheney

Previous studies have shown that knockdown of Rab5 increases the number of circulating hemocytes in Drosophila. The goal of this project is to explore the Rab5 protein and whether it acts through different receptors such as the domeless receptor of the Jak/Stat pathway or the Eater receptor of the Nimrod family. Domeless knockdown had little to no effect on hemocyte numbers while Eater knockdown led to an increase in hemocyte number comparable to Rab 5 knockdown.

DNA Mismatch Repair Gene Regulation from Oxidative Stress

Fred Zucule ’21; Advisor: Travis Brown

Mismatch repair (MMR) is a DNA repair pathway responsible for the recognition of DNA mismatches and the facilitation of their repair through recruitment of auxiliary proteins that excise and repair the mismatch. Defects in MMR cause an accumulation of DNA mismatches, which if left unrepaired can lead to genomic instability. MMR deficiency is mainly characterized by mutations in MSH2, MSH6 or MLH1 genes, which respectively code for factors that bind to mismatched base pairs. One of the most common metabolic disorders observed in humans is diabetes, where oxidative stress in cells is a primary consequence. Oxidative stress occurs when the concentration of reactive oxygen species (ROS) exceeds that of antioxidant species. ROS lead to mutations through the formation oxidative DNA adducts which cause DNA mismatches. While MMR is responsible for repairing these lesions, the regulation of MMR under oxidative stress is not well defined. We hypothesize that oxidative stress reduces MMR function through the downregulation of MMR gene expression. Here we show how the mRNA steady-state levels of MLH1, MSH2, and MSH6 are downregulated in HEK293T cells under oxidative stress. Understanding the regulation of MMR can elucidate how diabetes could increase the risk of cancer development.

CRP and Cra Have Opposing Activities in the Regulation of the Fructose Operon in Vibrio Cholerae

Christian Beck ’20 and Sayde Perry ‘22; Advisor: Jane Liu

Vibrio cholerae is the causative agent of cholera, a diarrheal disease which inflicts over 2.8 million individuals annually. A Gram-negative bacterium, V. cholerae survives in both the human small intestine, where pathogenesis occurs, and aquatic reservoirs, where the bacteria can spread and contaminate other water sources. In order to survive in both environments, V. cholerae must adapt to available carbon sources by selectively producing the necessary metabolic machinery. In this project, we focused our attention on fructose, one of the most abundant sugars in freshwater environments, and how V. cholerae regulates fructose metabolism at the genetic level. More specifically, we focused on the roles of Cra and CRP, two known transcriptional regulators in Escherichia coli, in regulating the expression of fruB, the first gene of the fructose operon fruBKA. Using assays to evaluate changes in expression at the transcriptional and translational levels, we found Cra to be a repressor of fruB and CRP to be an activator of fruB. We also found CRP to be a repressor of Cra, suggesting that CRP and Cra interact to affect downstream fruB expression. Using transcriptional fusions and mobility-shift assays, we will continue to tease apart the roles Cra and CRP play in regulating fructose metabolism, a process intrinsic to V. cholerae’s ability to thrive in two distinct environments.

Regulation of Vibrio Cholerae Mannitol Transporter by Proteins MtlR and CRP

Micayla George ’20; Advisor: Jane Liu

Cholera is a gastrointestinal disease caused by Vibrio cholerae that affects millions of people each year. V. cholerae is resilient in dynamic environments due to intricate cell signaling pathways that regulate intake of a variety of carbon sources including mannitol - an abundant carbon source produced by plants and algae. Mannitol intake by V. cholerae is dependent on the mannitol phosphotransferase system (PTS). The mannitol transporter protein is encoded by the mtlA gene located within a group of genes (mtlA, D, and R) on the mannitol locus; MtlA expression is repressed by protein MtlR and activated by cAMP receptor protein (CRP). It is known that CRP binds the mtlA promoter to activate transcription, but the mechanism of repression by MtlR is unknown. The aim of this project is to better understand the dynamics between MtlR and CRP protein regulators of the mtl locus to gain insight into MtlR regulation of MtlA expression. This was accomplished through assessment of MtlR and CRP levels across V. cholerae via Western Blots, as well as through assessing mtlA expression in the absence of CRP and/or MtlR via LacZ assays. Previous work has shown that MtlR levels fluctuate depending on available carbon source, but we found CRP levels to be relatively unaffected. We also found that while deletion of the mtlR gene increases mtlA expression, this is not observed when the crp gene is also deleted, suggesting that MtlR repression is secondary to CRP activation of mtlA expression.

Identifying CoADR and NPSR Function: A Metagenomic Analysis of the Sulfur Reducing Capabilities of CoADR and NPSR

Max Wragan ’22 and Archie Spindler ‘22; Advisor: Edward J. Crane

Coenzyme-A disulfide reductase (CoADR) and NADH-dependent persulfide reductase (Npsr) are enzymes that have long been suspected to metabolize inorganic sulfur compounds due to their anaerobic environments in which they are found. Previous studies have had difficulty proving that CoADR and Npsr perform sulfur respiration through direct approach, which is why this research explored CoADR and Npsr’s relationship with sulfur reduction indirectly through metagenomic analysis. These results compiled the geochemistry, biochemistry, and microbiology of a wide variety of proteins with known functions and explored their relationship with CoADR and Npsr. Our data was compiled through the Joint Genome Institute’s metagenome database and run utilizing their BLAST program. This data was analyzed through Principle Component Analysis (PCA) which suggested CoADR and Npsr’s role as sulfur reducing enzymes via their correlation and alignment with other known sulfur reducing proteins, and their lack of their of with sulfate reducing, oxidative stress, and reversible sulfur cycling proteins.

Identification of Novel Streptococcus Pyogenes Cas9 Functional Domains by CRISPR Tiling

Michelle Garcia ’22; Advisor: Travis Brown

The RNA-guided CRISPR-Cas9 system derived from Streptococcus pyogenes can be programmed to cut dsDNA containing a PAM sequence (NGG) for efficient and site-specific genome editing. Although the S. pyogenes Cas9 (SpCas9) protein has been structurally and biochemically characterized, its functional regions have never been explored in detail. Harnessing the RNA-mediated sequence-specific cleavage property of SpCas9, here we custom-built a tiling CRISPR library that targets every possible position at the SpCas9 coding sequence to interrogate its functionally important regions. Using red and green fluorescent protein reporters, we differentiated active SpCas9-expressing cells from the inactive SpCas9-expressing cells by fluorescence assisted cell sorting (FASC), and subsequently sequenced these two populations by Illumina second-generation DNA sequencing. By using in-house software to deconvolute the sequencing data, our CRISPR Tiling assay was shown to be able to identify previously known essential regions of SpCas9. Most importantly, our investigation revealed a potential new important domain of amino acids 418-427. We hypothesize this region conserves protein flexibility in the SpCas9 unbound apo-state as a pivot point for Helical domain reconfiguration upon binding to sgRNA. This hypothesis is tested by CRISPR-Cas9 targeting at evolutionarily conserved and CRISPR Tiling hot residues to validate our screen and purify mutant proteins for functional and structural profiling.

Analyzing RhoA Activity During Pancreas Development

Joana Rodriguez ‘21; Advisor: Travis Brown

The importance of the pancreas- an organ whose dual function is to produce enzymes for digestion and secrete hormones to regulate blood sugar homeostasis- is unmistakable. The most important of the diverse cells that constitute the pancreas is the endocrine beta cell, which secretes insulin and whose dysfunction is linked to diabetes.  Current stem cell technologies focus on differentiating these cells in vitro; however insufficient knowledge regarding the intracellular signaling pathways that lead to beta cell differentiation during normal pancreas development has been a significant limiting factor in the application and development of these technologies.  Studies regarding intestinal development have cited the RhoA GTPase signaling pathway, linked to general cell motility and cytoskeletal rearrangement, as essential for endocrine cell differentiation; however, its role in pancreas development remains unclear. Here, we investigate the dynamic regulation of the RhoA signaling pathway during pancreas embryogenesis using a mouse model organism. We adopted a “RhoA-FRET Biosensor Mouse”  in order to isolate the RhoA GFP-linked fluorescence to the pancreas, whose activity could then be determined based on its localization within the cell. Our preliminary findings suggest that RhoA exhibits a pattern of activation for the three major pancreatic cell regions. These results at different time points will allow us establish a pattern of RhoA activity during pancreas organogenesis.

RAD4 is Implicated in DNA Double Strand Break Repair

Waylon Henggeler ’20; Advisor: Cristina Negritto

DNA double strand breaks (DSBs) occur in the presence of ionizing radiation and can be extremely detrimental and even lethal to cells if repaired incorrectly. Homologous recombination (HR) is the most accurate method cells use to repair DSBs; during this process, the sister chromatid is used as a template to ensure that nucleotides are not lost during repair. HR is a complicated process that is not entirely understood and uses a variety of proteins. RAD50 is one such protein and is known to be crucial in DNA end resection, one of the initial steps in HR. This research seeks to illuminated whether RAD4, a protein crucial to the recognition steps of a different DNA repair pathway, nucleotide excision repair, is important for the recruitment of HR proteins to a DSB model in Saccharomyces Cerevisiae. A DSB was induced in three different strains: RAD4-FLAG WT, RAD50-FLAG WT, and RAD50-FLAG ∆rad4. Chromatin immunoprecipitation using anti-FLAG beads was then used in conjunction with quantitative PCR and western blots to investigate which proteins were recruited to the break site. We found that in wild type backgrounds RAD50 is clearly recruited to the break site, while it is unclear whether RAD4 is recruited. However, there is a significant difference between the recruitment of RAD50 in a WT background versus a ∆rad4 background. This suggests a novel function of RAD4: that it may be crucial in early DSB detection and the recruitment of other proteins to the break site.

Acetylcholine Signaling Affects the Route of Linker Cell Migration Through Muscarinic but Not Nicotinic Receptors

Jorge Gomez ’20; Advisor: Mihoko Kato

Acetylcholine receptors (AChR) are membrane proteins that respond to the neurotransmitter acetylcholine and are best known for their role in muscle contraction. Kato et al. found through transcriptional profiling that in the male Caenorhabditis elegans nematode, the linker cell (LC), a cell that leads the migration of the developing gonad towards the posterior body, expresses various neurotransmitter receptors including AChR. This research investigates the role that the two types of AChRs, nicotinic and muscarinic, have on facilitating the migration of this cell, the linker cell (LC). A knockout mutant of gar-3, the only muscarinic receptor expressed in the LC, was previously found to have mild defects in the LC migratory path. We investigated whether two of the multiple nicotinic AChRs expressed in the LC, acr-15 and acr-16, were also involved in gonad cell migration. The CRISPR-Cas9 ribonucleoprotein complex was utilized to create loss-of-function mutations in these genes. The effect of these knockdown mutations was studied through the imaging of the male gonad, to determine whether it formed the proper shape. Our data showed that the knockdown of receptor combinations resulted in no notable change to the migratory path of the male gonad. Due to the many nicotinic AChRs expressed by the LC, a future experiment would be to tag other candidate nicotinic receptors in the genome with green fluorescent protein to confirm LC expression and make more AChR mutant combinations.

Calcium Signaling Is Not a Downstream Effector of the GAR-3 Acetylcholine Receptor for Cell Migration

Eric Blair ’22; Advisor: Mihoko Kato

Acetylcholine (ACh) is a neurotransmitter expressed in many multicellular organisms. We studied its function during organogenesis in Caenorhabditis elegans, a transparent microscopic worm. During the 3rd and 4th larval stages of development of C. elegans, the cell that leads the migration of the gonad, the linker cell (LC), expresses a muscarinic ACh receptor – GAR-3 – in its plasma membrane. When GAR-3 is overactivated with extracellular ACh, the LC reverses its orientation which may cause migratory defects. To discover more about the molecular pathway connecting ACh reception and LC orientation we looked to calcium signaling, which is commonly associated with GAR-3 ACh reception in other cells. We generated a worm strain that expresses GCaMP in the LC, which is a fluorescent calcium indicator and increases its fluorescence in the presence of calcium ions. Using the drug aldicarb, which increases extracellular ACh and over-stimulates GAR-3, we found no increase in GCaMP fluorescence. This implies no increase in Ca+ signaling and suggests that Ca+ is not a downstream component of the ACh signaling pathway during gonadal migration. Further research directions include screening more GCaMP worms to confirm this finding and exploring the roles more signaling molecules like Ca+ have on LC migration.

Analyzing How Proteins PERM-2 and PERM-4 Contribute to the Structure and Function of the C.elegans Eggshell

Tessa Fujisaki ’21; Advisor: Sara Olson

Proteins are essential to the structure and function of cells. They assemble to build various structures such as eggshells. The purpose of this project is to use the nematode, C. elegans, as a model system to understand how proteins PERM-2 and PERM-4 create the impermeable outermost layer of the eggshell, the vitelline layer (VL). This project studied the VL’s means of assembly and impermeability.

First, SHuffle E. coli cells were inserted with plasmids containing PERM-2 and PERM-4 proteins. These plasmids were tagged with molecular molecular markers GST or 6Histine residue. The plasmids were verified by protein expression, purification through an affinity column and SDS-PAGE gels. Next, their solubility was tested in various concentrations of urea. Then, the newly soluble proteins were run through a native PAGE gel to determine how they polymerize individually and together in a non-denaturing environment.

The results concluded that the empty vector, GST::PERM-2 and GST::PERM-4 plasmids contained the correct vectors. 6Histine::PERM-2 did not contain the correct vector. Only GST::PERM-2 was found to be soluble in 6M urea. Native PAGE gel electrophoresis for PERM-2::GST was unsuccessful despite attempts to invert the electrodes and make different types of gel and various stain solutions. In the future, running a successful native PAGE gel for both proteins will allow for deeper analysis of how they polymerize and add to the function of the VL.

A Novel System for Engineering Homing Endonucleases

Fernando Bolio ’22; Advisor: Lenny M. Seligman

Homing endonucleases recognize and cleave specific long DNA sequences, and have been extensively used for genome editing. Previous work in the Seligman Lab and others demonstrated HEs could be engineered to target novel DNA sequences. The approach used involved changing specific regions of the protein known to contact DNA and screening these variants for the ability to interact with various DNA targets. In order to further engineer HEs, we are modifying an established system developed by David Liu’s Lab at Harvard known as Phage-Assisted Continuous Evolution (PACE). To accomplish this, we needed 2 different plasmids: an M13 phage derivative with the HE in place of M13 GeneIII, and a plasmid capable of inhibiting phage replication that can be inactivated by the desired HE. We have successfully created 2 different versions of the HE containing M13 phage derivatives as well as 2 different inhibitory plasmids using PCR and Gibson Assembly. We have also managed to create phage replicates of our M13 derivatives by using helper phage. We are currently testing different combinations of our plasmids to see if our modified PACE system can be used to engineer new HEs.

Knockout of ARX to Improve Pancreatic Beta Cell Differentiation

Martha Castro ’21

Type 1 diabetes, a chronic disease of little or no insulin production, affects millions of people worldwide. This condition is caused by loss of pancreatic beta (β) cells and leads to an inability to decrease bloodstream glucose levels. Transplantation of glucose-sensing, insulin-producing stem cell-derived β cells (SC-β) can provide a long-lasting treatment for type 1 diabetics. However, during directed differentiation towards beta cell lineage, other cell types such as SC-α, enterochromaffin, and exocrine are also generated. SC-α produced by the differentiation protocol exclusively express the ARX transcription factor. We hypothesized that knockout of ARX in human stem cells would decrease production of SC-α, thus increasing the yield of SC-β from the in vitro SC-β differentiation protocol. To test this, we performed flow cytometry assays on cells generated by the differentiation of ARX knockout stem cells. ARX knockout in human stem cells was achieved with CRISPR-Cas9. We observed that differentiation of ARX knockout cells resulted in the production of 5.35% SC-α and 13.85% SC-β, on average, as compared to 10.40% SC-α and 14.60% SC-β in wild-type cells. We concluded that knockout of ARX does not significantly increase the production of SC-β.

2017

Investigation of PERM-1 Localization in Oocytes and Embryos of C. elegans

Anne Berhe ’20; Advisor: Sara Olson; Collaborator: Norani Abilo ’20

The innermost layer of the nematode C. elegans eggshell, the permeability barrier, is one of the most impenetrable materials in the animal kingdom. However, not much is known about the composition or formation of this eggshell layer. C. elegans is a model organism for many parasitic nematodes and further research on this highly impermeable layer of the eggshell could lead to potential drug targets that could halt the viability of worms via the eggshell. Ascarosides, a lipid containing the sugar ascarylose and a fatty acid-like side chain, are predicted to be an essential component of the permeability barrier. Previous studies have revealed that PERM-1 is necessary for the formation of the permeability barrier and this protein’s epimerase and reductase domains are similar to those seen in the final catalyzation steps of ascarylose biosynthesis pathway. A primary goal of this project is to localize PERM-1 before and directly after fertilization. Our experiments this summer focused on analyzing colocalization patterns between PERM-1 and various organelles in the nematode’s oocytes and early embryos. PERM-1 had various levels of colocalization across organelles but did not consistently have high levels of colocalization in any one organelle.
Funding Provided By: Paul K. Richter and Evalyn E. Cook Richter Memorial Fund and The R. Nelson Smith ’38 and Corwin H. Hansch Fund for Summer Chemistry Research

Investigating the Half-site Reactivity of P. Horikoshii CoADR

Yongkang Zhang ’18; Advisor: Matthew Sazinsky; Collaborators: Angela Ling ’19, Maxim Leshchinskiy ’19, Liwam Nerayo ’20

Half-site reactivity is an enzyme characteristic in which the active sites of a multimeric enzyme “fire” alternatively. At any given time, only one-half of the identical enzyme subunits are active. Although many metabolically important enzymes with half-site reactivity have been identified and characterized, the cause of half-site reactivity and its significance on enzymatic catalytic efficiency and mechanisms remain unclear. In this project, we designed, expressed and purified an asymmetrical dimer for P. Horikoshii Coenzyme A disulfide reductase (phCoADR), a member of the pyridine nucleotide disulfide oxidoreductase (PNDORs) family that has shown to display half-site reactivity during in vitro elemental sulfur reduction. Preliminary kinetics data suggest that the asymmetrical dimer loses the ability to fire alternatively. Preliminary data from NADH anaerobic titration of the enzyme reveal a difference in reduction potentials between the active and dead subunit of the enzyme.
Funding Provided By: The R. Nelson Smith ’38 and Corwin H. Hansch Fund for Summer Chemistry Research

Mutating GDI in Drosophila Using CRISPRCas9

Dominique Bruncko ’18; Advisor: Clarissa Cheney

The Rab pathway is extremely important for regulating vesicle transport. Rabs are G-Proteins, meaning that they act as a switch based on whether GTP or GDP is bound. One protein that helps regulate this switch is called a guanine nucleotide dissociation inhibitor (GDI), which holds the Rab in its GDP bound off state and covers its hydrophobic tail to transport it through the cytoplasm. GDI is N-terminally acetelyated post translation by NAT-B based on GDI’s second residue being Asn. Our objective is to mutate the second residue of GDI from Asn to Ser to see how being a substrate of NAT-A instead of NAT-B will change GDI’s interaction with Rabs. We are doing this by creating a Drosophila mutant with the mutated GDI in its genome via the CRISPR-Cas9 system. This system uses a protein to cut in a highly specific location, cleaving out the unwanted gene segment. Cells are then injected with a plasmid containing the Ser mutation as well as regions of homology to the genome to introduce the mutation via homologous recombination. Currently, the CRISPR-Cas9 system and its associated gRNAs have been prepared, and the homology plasmid is in the process of being created by using Gibson Assembly. The first several attempts at assembly were not successful, so several changes were made to optimize assembly in the future. The next step after the creation of the homology plasmid is to send the gRNA and the homology plasmid to be injected into our CRISPR-Cas9 Drosophila strain.
Funding Provided By: The R. Nelson Smith ’38 and Corwin H. Hansch Fund for Summer Chemistry Research

Cause and Effect of Natural Codon Reassignments: eRF1 Structure and Tandem Stop Codons in Ciliates

Ira Fleming ’18; Advisor: Andre Cavalcanti

Tandem stop codons, used as a safety-net in the event of codon readthrough, are found to be highly over-represented at the +2 position alone or the +3 and +4 positions together after the annotated stop codon in several ciliate species as well as in drosophila melanogaster, the latter known for its high rates of stop codon readthrough. The reassignment of stop codons in many of these ciliate species and the speculated reduction in termination efficiency, hinted at by higher levels of tandem stops, may be related to several identified point mutations within and adjacent highly conserved codon recognition sites and the GGQ’ peptide release site within eukaryotic release factor one.
Funding Provided By: The Professors Corwin Hansch and Bruce Telzer Undergraduate Research Fund

Protein Complexes that are Scaffolded by a Common Protein and Regulate C. elegans Eggshell Formation Localize Independently

Julian Prieto ’20; Advisor: Sara Olson

In the nematode C. elegans, fertilization of an oocyte leads to the hierarchical development of different eggshell layers and a reorganization of the embryo that serves to protect the embryo from polyspermy and assure its development. The vitelline layer, the outermost layer of the eggshell, is present on oocytes and separates from the plasma membrane immediately after sperm entry. Very little is known about the mechanics and composition of this layer, but CBD-1 (chitin-binding domain protein) has been identified as essential for this point of embryonic development. Through both RNAi and CRISPR experiments, PERM-2 and PERM-4 were identified as proteins having a co-dependent function essential in the formation of the vitelline layer. CBD-1 scaffolds both the PERM complex and the EGG complex (EGG-1-5, which is required for fertilization and egg activation).  By using CRISPR null mutants and domain mutants, I showed that the localization of each of these protein complexes are independent of each other. Interestingly, some of the perm-2(null) mutants still form some viable eggshells.
Funding Provided By: The Professors Corwin Hansch and Bruce Telzer Undergraduate Research Fund

The Roles of Rad50 and TFIIH in Double Strand Break Repair in saccharomyces cerevisiae

Robert Buxton ’18; Advisor: Tina Negritto

The transcription factor TFIIH has long been theorized to play an important role in the repair of double strand breaks (DSBs) in DNA. In this study, DSBs were induced in various Saccharomyces cerevisiae strains displaying both wild types (WT) alleles and mutations in either the Rad50 or Rad3 gene, and samples were taken of each strain at various time points. Either the Rad50 protein or the TFB1 component of the TFIIH transcription factor was tagged with FLAG peptide tags. The DNA associated with the tagged protein was then isolated via chromatin immunoprecipitation (CHIP) and quantified via QPCR to analyze the degree of occupancy of the target protein at the DSB at various time points in both WT and mutant yeast. In both TFB1 and Rad50 pull down, primer choice was found to play a large role in determining the magnitude of the degree of occupancy, but the shape of the curve was preserved. Rad50 pull down at the first of two recruitment events was substantially decreased in the Rad3-G595R mutant but was not completely knocked down, hinting at a potential role of TFIIH in facilitating recruitment of Rad50 to the DSB. The Rad3 mutant does not display the second of two increases in occupancy shown by the WT strain, showing that TFIIH may be required for the second recruitment of Rad50 to the DSB but not the first. An assay must be designed to determine whether increases in occupancy are due specifically to the behavior of the protein of interest at the experimental region or the primer region.
Funding Provided By: The Professors Corwin Hansch and Bruce Telzer Undergraduate Research Fund

The Characterization of rad3 Mutant Phenotypes in Nucleotide Excision Repair

Zoe Zhou ’18; Advisor: Tina Negritto

UV radiation can cause covalent linkages to form between adjacent DNA base pairs. Normally, this type of damage is repaired by a process called Nucleotide Excision Repair (NER). However, in a number of human diseases, including trichothiodystrophy (TTD), mutations in the genes encoding NER proteins prevent DNA repair from occurring. In some cases of TTD, this confers a photosensitive phenotype. For this study, we want to characterize the phenotypes of four different NER mutants, rad4, rad3-G595R, rad3-R660C, and rad3-A596P in S. cerevisiae. The rad3 mutants were created to mimic known mutations in human TTD patients. During NER, Rad4 plays the role of recognizing UV-induced DNA damage, and Rad3 acts as a DNA helicase that unwinds the DNA and recruits subsequent repair proteins. To characterize the mutants, a pinning assay and pulsed-field gel electrophoresis (PFGE) assay were used. For the pinning assay, cells were exposed to four different levels of UV radiation: 0, 50, 75, or 100 J/m2 and then grown over two days. For the PFGE assay, time points were collected after treatment with UV. Samples were subsequently treated with either mung bean nuclease or TUNEL staining to detect formation of single-stranded breaks after thymine dimer excision. The results from these assays suggest the mutant strains behave differently from the wild-type and also behave differently from one another. These differences may explain the underlying molecular defects in DNA repair that lead to TTD.
Funding Provided By: The Doudna and Cate Chemistry, Biology and Molecular Biology Fund, The Doudna and Cate Chemistry Fund

PERM-1 ligand screening through Differential Scanning Fluorimetry

Inga Van Buren ’18; Advisor: Matthew Sazinsky

Parasitic nematode infections pose a major worldwide health problem affecting more than 2 billion individuals each year. Current treatments, however, are specific for worms in the larval stage as embryos are protected by a permeability barrier. Previous studies have identified PERM-1 as a transmembrane protein implicated in the synthesis of the permeability barrier in C. elegans, a model organism for nematode infections. The goal of this project was to utilize differential scanning fluorimetry (DSF) to better characterize the binding partners of PERM-1, serving as a starting point for future drug screening. A truncated recombinant construct was expressed in a Shuffle cell overexpression system and purified using a detergent-based protocol. DSF was then used to tentatively assess the extent to which PERM-1 was folded, as well as its ability to bind NAD+, CDP, and UDP. Preliminary results suggest that PERM-1 has a melting temperature of ~53C and exhibits a thermal shift upon substrate exposure. However, observed thermal shifts are not consistent and could stem from the inherent instability of PERM-1 after purification. As a result, buffer, dye, and protein concentrations used in DSF must be further optimized to enhance protein stability and its behavior.
Funding Provided By: HHMI

Experimental Examination of C. elegans Ovulation Differences in Wild Type and trp-3 Mutants Through Live Imaging Microscopy

Katiannah Moise ’18; Advisor: Sara Olson

C. elegans has been used as model organisms to better understand fertilization and egg development. Easy view of meiotic division of oocytes without dissection has been helpful in developing the field around in vivo egg development. Egg activation is prompted by biochemical reactions. Calcium signals in the early stages of oocyte fertilization facilitates the transition from egg to embryo. This egg activation includes the formation of the eggshell that prevents polyspermy and allows for reactivation of the cell cycle. Calcium signaling has been modeled as a two-step process. The first step is an initial spike when the egg and sperm fuse, and the second is the propagation of a slow wave of calcium throughout the length of the egg. Takayama & Onami (2016) observed that the initial Ca2+ spike is produced by a sperm-specific Ca2+-permeable transient receptor potential (TRP)-3 channel that is delivered to the egg upon fusion. Without it, the initial spike is missing, but there is still a delayed calcium wave propagation. Not much is known about different sources of calcium channels that participate in the event, so I am examining the role of the egg’s ER in providing calcium for the slow wave. In addition, I am using live imaging microscopy to visualize ovulation and calcium wave differences between trp-3 mutant and wild-type embryos. The data will be used by collaborators in the math department to develop better 1D/2D mathematical models to describe the calcium wave propagation.
Funding Provided By: J. Steller Summer Curriculum Enrichment Fund

Engineering riboswitch-based whole cell biosensors through dual genetic selections and fluorescence activated cell sorting

Maryann Zhao ’18; Advisor: Jane Liu

Whole-cell biosensors are tools used to assess the bioavailability and bioaccessibility of small molecules. Compared to traditional instruments, biosensors offer the advantage of not only being easy and cheap to replicate but also requiring minimal specialized expertise. Riboswitches, nature’s own versions of biosensors, are well suited for this purpose because of their molecular recognition capabilities. Generally found in the 5’-untranslated region of prokaryotic mRNA, riboswitches are genetic regulatory elements that switch between conformational states in the presence of a small molecule ligand. The binding of the small molecule to the mRNA aptamer domain induces structural changes in the expression platform that result in transcriptional and/or translational modulation of gene expression. My project harnessed riboswitch’s natural mechanism to answer the question, is it possible to engineer an artificial riboswitch that can sense a selected small molecule and control gene expression in a characteristic manner? Using dopamine as the small molecule, our platform used libraries of Escherichia coli, each harboring a mutagenized variant of a natural riboswitch, and performed dual genetic selection and fluorescence activated cell sorting to isolate functional riboswitches. Developing an effective platform to engineer riboswitch-based biosensors will provide researchers with a powerful detection tool that can be applied in diverse situations.
Funding Provided By: Department Funding

Investigating a Functional Role for N-terminal Acetylation of Rab GDI in Drosophila

Michael Poeschla ’18; Advisor: Clarissa Cheney

Rab GTPases comprise a large family of proteins that regulate a variety of membrane trafficking processes related to vesicular transport. 1 GDI is an essential part of the Rab pathway, responsible for recycling inactivated Rabs from membranes back into the cytosol. 2 Previous data from the Cheney laboratory has shown that GDI is N-terminally acetylated in Drosophila, and that mutations in the acetyltransferase that catalyzes this modification are lethal in the late larval (3rd instar) stage. 3 Though over 80% of eukaryotic proteins have been found to be N-terminally acetylated, the functional consequences of this modification are poorly understood. 4 The aims of this study are to construct an in-vitro system by which we can assess the effects of N-terminal acetylation of Rab GDI on its ability to bind and extract Rabs from membranes.
Funding Provided By: HHMI

Using FUCCI to investigate Mitotic Activity in Rab5 knockdown Hemocytes and Imaginal Wing Discs in Drosophila

Priyanka Ramanan ’19; Advisor: Clarissa Cheney

Rabs are G-proteins that are important directors of vesicle traffic in the cell. Previous studies demonstrate that knockdown of Rab5 in the hemocytes of Drosophila results in an overabundance of circulating hemocytes. Knockdown of Rab5 in the imaginal wing discs also results in malformed wings. The goal of this project was to use the FUCCI system to explore cell over proliferation as a possible explanation. FUCCI transgenes were introduced to specifically be expressed in the imaginal wing disc tissue and in hemocytes in order to visualize the cell cycle. No significant difference was found in mitotic activity between wild-type and Rab5 knockdown imaginal wing disc and circulating hemocytes.
Funding Provided By: J. Steller Summer Curriculum Enrichment Fund

Identifying MtlS Targets in Vibrio cholerae

Sabrina Mendez-Contreras ’18; Advisor: Jane Liu

MtlS is a small RNA transcribed antisense to the 5’ untranslated region of mtlA, which codes for the sugar transporter for mannitol. MtlS works in cis as a translational repressor of mtlA. Because bacterial sRNAs can target multiple genes, we hypothesized that MtlS may regulate other genes in addition to mtlA. We used RNA target-prediction programs and mass spectrometry-based proteomics to identify additional MtlS targets. The results identified 6 potential targets, including mtlA. To confirm that MtlS is regulating the predicted targets, we created translational fusions of each target with gfp and introduced these constructs into V. cholerae strains with and without mtlS. This allowed us to monitor the effects of mtlS expression on fluorescence levels, and thus the amount of target-gfp being expressed. The results were also confirmed by western blot analysis. As a positive control, an mtlA-GFP fusion was constructed and I observed the expected down-regulation. I observed stationary phase down-regulation of VC1899 in the presence of MtlS, contrary to the up-regulation indicated by the proteomics results. Moving forward, I will determine where MtlS is binding VC1899 transcript to affect down-regulation of the gene. Additional putative targets of MtlS are also in the process of being tested. Ultimately, this work will expand our knowledge on MtlS’s role in V. cholerae and sRNAs in general.
Funding Provided By: Department Funding

Evolution of RNA-based biosensors in E. coli yields riboswitches with over three-fold activation

Samuel Lin ’20; Advisor: Jane Liu

Small molecules play important roles in cell metabolism and function; thus, developing efficient small-molecule sensors is integral to our further understanding of cell functions with many potential applications. In order to create a small-molecule sensor, we created a RNA-based biosensor constituted of two main components: in the 5’ untranslated region, an aptamer domain that binds the ligand of interest, and between the aptamer domain and the Shine-Dalgarno (SD) Sequence, an expression platform that mediates conformational changes of the mRNA, which upon ligand binding, causes a change in downstream gene expression. For use as a biosensor, gfp was placed downstream of the SD sequence, and thus cell fluorescence would be turned “on” and “off” following ligand binding. Also placed downstream of the SD sequence is the gene tetA, which allows for genetic selection of riboswitches from a randomized RNA library. In order to evolve the Liu Lab’s previously found theophylline riboswitch, the aptamer domain of the initial “hit” was mutagenized, thus creating a 107 library of E. coli, each harboring a unique RNA library member on which to perform genetic selections and fluorescence-activated cell sorting (FACS). Three rounds of selections on the mutagenized library yielded riboswitches exhibiting over three-fold activation upon ligand binding. Refinement of our selection and screening protocol may lead to discovery of riboswitches with even higher activation and overall fluorescence.
Funding Provided By: Department Funding

Clinically Tested NDV-3A Vaccine May Protect Against Highly Virulent Multi-drug Resistant Fungal Infection

Zhaoqi Huang ’19; Advisor: Jonathan Wright

C. auris is an emerging opportunistic fungus that has recently presented itself as an enormous threat in hospital settings around the world. Of the few infectious species of candida, C. auris has gained attention because of its growing resistance to all three major classes of antifungal drugs. In immunocompromised patients, the fungus can enter the bloodstream and cause invasive candidiasis, affecting the central nervous system, organs, and may lead to death. The current mortality rate for patients infected with C. auris is estimated to be 30-60%. Candida albican’s Als3 protein based NDV-3A vaccine has shown protection against C. albican infections in preclinical and clinical trials (phase I/IIb). Through bioinformatics, we found that C. auris contains a cell surface protein that shows amino acid homology to the Als3 protein. Using various molecular biology and immunodetection tools, we identified and isolated the Als3 protein homologue in various C. auris strains. This finding has serious implications in Candida vaccine development as previously tested NDV-3 vaccine might show protection against highly virulent and drug resistant strains of Candida auris.
Funding Provided By: Department Funding