Managing cultures is a vital task in synthetic biology, but constantly measuring and adjusting culture conditions is both tedious and labor intensive. Modern assays that accomplish this, such as High Precision Liquid Chromatography, can also be prohibitively expensive. Our project aims to reduce the amount of time and effort needed to maintain cultures through the creation of an affordable image analysis system that autonomously reads visual data to measure the current state of a culture and then determines whether to release inducer chemicals based on user input. Our project utilizes the violacein pathway to simulate other metabolic pathways by producing four colored signals dependant on a two gene switch. These outputs are then measured by an open-sourced Raspberry Pi (a small, standalone computer) setup, which captures visual data via camera, measures the culture’s RGB value, and then directs the gradual release of inducer chemicals to maintain or change the culture’s color over time. Currently, yeast strains capable of coexpressing both a violacein and a non-visible pathway are being cloned. In addition to this, a prototype containing 3D-printed syringe pumps, a chemostat, and image analysis software in development. By combining both biological and hardware systems, we expect our unique design to be able to generate previously unavailable visual data regarding certain biosynthesis processes, such as those involved in antibiotic production and fermentation.

Even though Breast Cancer type 1 gene (BRCA1) was the first gene to be definitively linked to hereditary breast and ovarian cancer, much is still unknown about how BRCA1 influences cancer risk. What is known is that certain BRCA1 variants confer an increased risk of early onset breast, ovarian, prostate and peritoneal cancer, but there are also many patients with variants of unknown significance that have yet to be characterized. Current methods for variant characterization require arduous genetic manipulations of mammalian cell lines, which could be simplified if a homologous model organism was used. A BRCA1 homolog has been identified in the nematode, Caenorhabditis elegans, indicating that the nematode could be a useful BRCA1 model system. My preliminary results indicate that activity of BRCA1, the gene product of BRCA1, is conserved in the worm homolog as both proteins expressed functionally homologous ubiquitin-ligase activity when studied using purified components. In order to investigate the conserved residues in the C. elegans homolog that lead to the conserved activity, I am comparing the interactions of both BRCA1 homologs with the E2 enzyme (required for ubiquitin-ligase activity). Using site-directed mutagenesis, I am producing mutations at the hypothesized E2 binding site on the worm homolog that I predict will inhibit ubiquitin-ligase activity by preventing E2 binding. The impact of these mutations are being studied through biochemical ubiquitin-ligase assays using purified components. A nematode-based BRCA1 model would allow for hypotheses developed through in vitro or in cellulo studies to be tested in a whole organism, significantly expediting variant characterization. Understanding the importance of the conserved residues between the homologs will identify key regions of BRCA1 where patient variations may be more likely to pose a risk.

Posttranslational modification with the small protein ubiquitin (Ub) is an universal and essential process in various regulatory pathways of eukaryotes (from yeast to humans), ranging from protein degradation to DNA repair. Misregulation of ubiquitination is associated with diseases such as cancer and neurodegenerative disorders. Ubiquitination is accomplished by an enzymatic cascade that involves a family of ~40 E2 conjugating enzymes, which form high-energy thioester (R–S–CO–R) intermediates with Ub, called E2~Ub conjugates. For the majority of ubiquitination reactions, it is the E2 that covalently attaches Ub to substrate proteins. While the overwhelming majority of E2s ubiquitinate the ε-amino group of substrates’ lysine residues, important exceptions have recently been discovered. For example, Ube2W has been identified as the only human E2 that attaches Ub exclusively to the ɑ-amino group of substrates’ unstructured N-termini. Knockout of Ube2W in mice has lead to severe developmental abnormalities, suggesting it is important for early postnatal survival. Little is known about the molecular mechanism of Ube2W’s unusual specificity and structural insights to the catalytically-relevant species, the Ube2W~Ub conjugate, are lacking. Through mutational analysis and biochemical assays, I seek to understand the following question: What role does Ube2W’s capability to dimerize and its unusually high number of cysteine residues play in its catalytic mechanism? In addition, I am using a combination of chemical biology and nuclear magnetic resonance (NMR) to study the Ube2W~Ub conjugate to track structural changes at the E2’s active site during catalysis. This investigation will provide insight into biochemically fascinating and biologically highly relevant enzyme.

Living organisms are composed of cells; thus, cells are called the basic building blocks of life. In order for an organism to grow, cells undergo mitosis. During mitosis, chromosomal DNA is replicated and segregated into two daughter cells. Errors in segregation can lead to the development of some forms of cancer. Because of this, equal chromosome segregation is a critical mitotic process. Microtubules are fibril-like cytoskeletal structures that are responsible for chromosome separation. They originate from two centrosomes. The growth and extension of microtubules push the two centrosomes apart, creating two poles in a mitotic cell. Kinetochores bind to these microtubules and align at the center of the cell. Disassembly of these microtubules is coupled to the equal segregation of sister chromatids. In Saccharomyces cerevisiae, correct kinetochore-microtubule attachment is mediated by the Ndc80 and Dam1 complexes. The heterotetrameric and rod-like Ndc80 complex is the main microtubule-binding component of the kinetochore. The heterodecameric Dam1 complex forms oligomeric rings around microtubules and enhances the microtubule-binding capacity of the Ndc80 complex. In vitro, the Ndc80 complex bridges two Dam1 complex rings, yielding a specific 33 nm inter-ring distance. To investigate the importance of this distance, we constructed various mutant Ndc80 complexes to further increase the Dam1 complex inter-ring distance. Specifically, each Ndc80 complex mutant contained an 8, 10, or 12 heptad repeat insertion that elongated the length of the coiled-coil domain of the Ndc80 complex by 8, 10, and 12 nm, respectively. We tested if these mutant Ndc80 complexes support cell division by transforming a yeast strain that does not contain wild-type Ndc80 gene and selecting for growth on SD-ura low adenine plates. These produced non-sectoring red colonies; therefore, the constructs did not support cell division. This suggests the specific 33 nm Dam1 complex inter-ring distance is important for cell viability.

Aggressive cancers are resistant to apoptosis, and stem-like cancer cells might be responsible for re-growth of cancerous tumors. This inspires the search for compounds that could aid in the destruction of cancer cells with stem-like properties. Drosophila germ line stem cells under radiative or chemo-toxic stress are protected by a molecular signal from apoptotic daughter cells via the TIE receptor. Thwarting this protective signal by the introduction of a small molecule may lead to the discovery of compounds that can be effective against stem-like cancer cells. The National Cancer Institute (NCI) diversity set of 1,596 small molecules is screened, in vivo, to find compounds that can induce apoptosis in the germ-line stem cells of Drosophila melanogaster. We tested several compounds from the NCI diversity set on 0-5 day-old fruit flies to get an understanding of the effects of the compounds on stem cells. Multiple compounds, including Camptothecin and Maytansinol, have been found that can induce apoptosis in both the male and female germ-line stem cells. Future investigations will uncover the mechanism of action and efficacy against stem-like cancer cells. After determining which compounds work effectively to target stem cells, this knowledge will be applied to understanding the mechanism responsible for resistance to cell death by stem cells and potentially some cancer cells, which could lead to more effective cancer-eradicating drugs.

Enteric diseases, or diseases of the gastrointestinal (GI) tract, remain one of the most prevalent killers of children in sub-Saharan Africa. The most practical way to prevent such diseases is through vaccination, but antigens for enteric diseases need to be delivered directly to the GI tract to be most efficient, making vaccination difficult. Recent studies by the von Adrian group at Harvard University have found that certain kinds of white blood cells are reprogrammed to home to the GI tract when they encounter retinoic acid, commonly referred to as vitamin A. The King group at the University of Washington is working to develop a vaccine using recently developed protein cages, which can transport all-trans retinoic acid (ATRA) and enteric antigens. The first step to developing this vaccine is to create a hyper-stable base protein for the cage, to allow the protein to be engineered into a protein cage. To do this, I created 23 different versions of Cellular Retinoic Acid Binding Protein I (CRABP-I) each with a different combination of 5 mutations. These mutations were identified using the PROSS server from the Weissman Institute which uses Rosetta to identify mutations which will stabilize a given protein. These constructs were then tested for stability at increasing temperatures. The construct that resisted denaturing at the highest temperature was selected, and I further mutated the protein to help it bind ATRA. This protein will be assembled into a cage combining ATRA and a model antigen, and sent with several other controls for testing in mice. We hope that our protein cage vaccine will generate increased immune responses in mice against enteric diseases.

Duchenne Muscular Dystrophy (DMD) is an X linked, recessive genetic disease caused by mutations in the dystrophin gene. The disease is characterized by progressive muscle degeneration. Dystrophic mice (mdx^4cv) are a DMD animal model. Here, we compared mdx^4cv and WT mice to quantitatively assess motor performance. We hypothesized that the mdx^4cv mice would show differences in gait compared to WT as a result of muscle weakening. To test our hypothesis, mice were assessed at 1.5, 3 and 6 months of age using the Noldus CatWalk XT system to analyze gait. The mice voluntarily moved across an enclosed walkway on a backlit glass plate. The prints were captured by a camera and later analyzed using the CatWalk software. Our results show that there are significant differences in gait between the WT and mdx^4cv mice. The mdx^4cv mice were slower to cross the runway than WT at 1.5 (mdx^4cv: 3.60.8s WT: 2.80.5s) and 3 months (mdx^4cv: 3.80.9s, WT 2.51.2s ). The distance between the front paws of the mdx^4cv mice were wider than WT at 1.5 months (mdx^4cv: 3.60.8cm WT: 2.80.5cm), 3 months (mdx^4cv: 3.80.9cm WT: 2.51.2cm) and 6 months (mdx^4cv: 1.50.2cm WT: 1.20.2cm). Print position is the distance between the position of the hind paw and the position of the previously placed front paw on the same side. In mdx^4cv mice the print position was larger than WT at 1.5 (mdx^4cv: 1.30.8cm WT: 0.40.2cm) and 3 months (mdx^4cv: 1.40.8cm WT: 0.50.4cm), indicating that the hind leg moved a shorter distance in a single stride. The gait analysis assay can be extended to assess gene therapy based treatments that our lab is currently developing. This will allow us to determine if the treatments are successfully able to mitigate the effects of DMD.

As cancer remains a looming threat to the public, understanding the genetic and biochemical basis of how cancers operate is necessary in order to develop new medicines and treatments. A topic of critical importance when considering the biochemistry of cancers is how specific changes in gene expression control the growth of tumors. I explore this topic by using Drosophila genetics in order to investigate the crosstalk amongst three key growth regulators, RAC-alpha serine/threonine-protein kinase (Akt1), Translationally Controlled Tumor Protein (TCTP), and Yorkie (Yki). The overexpression of Yki in intestinal cells is known to cause intestinal tumors, and our preliminary results indicate that Akt1, TCTP, and Yki interact to control intestinal stem cell proliferation. Nevertheless, signaling crosstalk amongst these three genes is still ambiguous. By using the advantageous genetic toolbox of Drosophila, I created experimental fly lines which allow me to manipulate the expression of each gene or a combination of these genes in Drosophila intestinal stem cells. My goal in this project is to anatomize this signaling crosstalk through monitoring how manipulation of these genes in combinations influence the proliferation of intestinal stem cells using confocal microscopy. Ultimately, we hope to attain a greater understanding of growth in relation to genetic networks that control it.

During development, the repression of transcription factor tbx16 allows embryos to dynamically allocate neural and muscle tissue in the correction proportion as the body axis is formed. Understanding how tbx16 is regulated is crucial for identifying the underlying mechanisms of neuromuscular developmental defects. Currently, we are investigating how two genes of interest termed eve1 and vent interact to regulate the expression of tbx16 in zebrafish embryogenesis. Previous studies have relied on Morpholino anti-sense oligonucleotides (MOs), but MOs often create off-target effects and misleading phenotypes that don't reflect genetic mutants. Using the CRISPR-Cas9 system in zebrafish, we have created true genetic knockouts for eve1 and vent. By analyzing the difference in phenotypes between eve1 mutants, vent mutants, and eve 1/vent double mutants, we can determine if eve1 and vent act redundantly to repress tbx16. Our goal is to uncover the mechanisms behind these mutant phenotypes in order to identify the role that eve1 and vent play during embryogenesis. Our research aims to further our understanding about how serious birth deficits arise from gene deficiencies and complications in development.

Proteins serve very specific and powerful functions in the cell by maintaining a specific shape through chemical interactions in the protein sequence. Under stress conditions however, these chemical interactions holding the protein together may break apart, leading to the protein’s loss of function and exposure of hydrophobic patches that can interact with other proteins to form toxic aggregates. Heat shock proteins (sHSPs) act as safeguards under conditions of stress and protein misfolding as molecular chaperones that interact with exposed hydrophobic patches to stabilize unfolded proteins. The importance of sHSPs in the cell is exemplified through various disease associated mutations of sHSPs. sHSPs are a large and diverse family of proteins distinctive in their ability to form multimers of various sizes, which correlates with varying ability to prevent aggregation. However, little is known about how different structural elements of sHSPs influence function. The Klevit lab aims to better understand the molecular interactions of human sHSPs with themselves, how these sHSPs are activated in cellular conditions, and how they recognize and bind unfolded protein. My work focuses on the characterization of the structure and function of the human sHSP, HSPB8. Specifically, I am studying the effects of HSPB8 binding to zinc, the cellular levels of which vary in response to stress and regulate the function of many other proteins. By comparing zinc bound and non-zinc bound protein samples using various biophysical techniques such as NMR, circular dichroism and fluorescence, I aim to elucidate the effects of zinc binding on structural and functional elements of HSPB8.

With the rise of antibiotic resistant bacteria, understanding how pathogenic bacteria invade and survive within our bodies is becoming more important as we search for novel ways to combat bacterial infection. Some pathogens, such as Salmonella typhimurium, inject host cells with proteins that suppress the host cell’s ability to respond to bacterial invasion. One such protein, SspH1, is an E3 ubiquitin ligase. E3 ubiquitin ligases attach ubiquitin, an important regulatory protein used to mark other proteins for degradation, to a substrate. Ubiquitin, the biochemical machinery that activates ubiquitin, and the pathways that utilize ubiquitin as a signaling molecule, are not found in bacteria, only in eukaryotes. However, bacteria have evolved proteins, such as SspH1, that hijack a host cell’s biochemical machinery in order to target host proteins for degradation. SspH1 consists of two domains: a catalytic E3 domain that transfers ubiquitin to substrate, and a substrate recognizing Leucine-Rich-Repeat (LRR) domain. I have shown through biochemical assays that the LRR domain inhibits the catalytic activity of the E3 domain. I have also shown that this inhibition is relieved in the presence of substrate. Through the use of 2D NMR and further biochemical assays, I hope to characterize the binding between the LRR and E3 domain in order to further the understanding of how SspH1 is inhibited. Understanding the mechanism of SspH1 auto-inhibition can increase our knowledge of the mechanisms and activities of related bacterial effector proteins, furthering our understanding of how pathogenic bacteria evade our immune system, and potentially lead to the development of novel treatments that can combat bacterial infection.

Stressed cells may be able to avoid elimination but continue to respond to compensatory growth signals and accumulate, resulting in tumor formation. This ability to avoid cell elimination processes, such as apoptosis, is one of the hallmarks of cancer. The Jun N-terminal Kinase (JNK) mediates cellular stress responses by inducing apoptosis of stressed cells and promoting compensatory cell proliferation. Using immunohistochemistry and the GAL4-UAS targeted gene expression system, we are able to distinguish intestinal stem cells (ISCs) from enteroblasts, enterocytes, and other intestinal cells through a confocal microscope. We found that activation of JNK in fruit fly intestinal stem cells (ISCs) induced temporary over-proliferation. On the other hand, persistent activation of JNK resulted in elimination of the ISCs. Strikingly, we discovered that ISCs with constitutive activation of JNK 'extrude' out from the epithelial layer of fly intestines. Our findings suggest that cell extrusion may be a mechanism for JNK-mediated tumor suppression, preventing the accumulation of cancerous cells. Alternatively, cell extrusion may be involved in metastasis, enabling cancerous cells to spread to other parts of the body. We aim to further investigate the molecular mechanisms and gene expression underlying cell extrusion in various genotypic backgrounds, which may be helpful to understand how cancerous cells evade cell elimination and undergo metastasis. 

  Protein homooligomers, multibodied assemblies built from identical polypeptide chains, comprise a large fraction of known cellular proteins. Homooligomers prove to be particularly amenable for many biological applications; they hold the potential as oligomerization domains, often have enzymatic functionality as a byproduct of their oligomeric configuration, and can serve as structural scaffolds for bionanomaterials. While there exists a multitude of protein homooligomers in the Protein Data Bank, the finite number of existing homooligomers limits the potential for custom applications. Our current work involves designing novel cyclic protein homooligomers from a set of de novo designed repeat proteins that bind the Fc region of antibodies. Using the Rosetta software suite, we generated a set of de novo homooligomer models by designing the oligomeric interface to direct self-assembly into a target configuration with three to six identical chains. After a round of refinement, we will express the designs in Escherichia coli and purify them by immobilized metal affinity chromatography. Their oligomerization state will be validated by measuring the molecular weight in solution by size exclusion chromatography paired with multi-angled light scattering and comparing it to the predicted molecular weight of the design. Designs that exhibit the desired molecular weight will be submitted to collaborators for small angle X-ray scattering data and X-ray crystallography. The exclusive use of de novo proteins in homooligomer design grants a greater control over the shape and stability by nature of the repeats, thus making one successful interface design useful for a multitude of shapes and sizes. This variability opens up a wide scope of scaffolds for using these homooligomers for near atomic scale structural characterization by cryo-EM.

Our laboratory has identified a group of neurons that expresses calcitonin gene-related peptide (CGRP) in the parabrachial nucleus (PBN), which are involved in appetite suppression and responses to fear. For example, CGRPPBN neurons mediate the suppression of food intake during a conditioned taste aversion (CTA) and are activated following conditioned fear responses [1,2,3]. Here, we investigate whether CGRPPBN neurons mediate anorexia associated with a diet deficient in essential amino acids (EAA-def anorexia). We inactivated CGRPPBN neurons by injecting an associated adenovirus (AAV) carrying the tetanus toxin light chain (Tet tox) in the PBN of CalcaCre/+ mice (Calca is the CGRP gene) and showed increased food intake and decreased body weight loss compared to control animals on a prolonged diet deficient in EAAs. In a pair fed group of mice injected with the Tet tox virus, there was not a significant difference in body weight loss. These results suggest that CGRPPBN neurons modulate appetite during EAA-def anorexia, but are not responsible for a change in energy homeostasis during EAA-def anorexia.

The forefront of modern medical research involves decoding the biological pathways that make disease transmission and perpetuation possible. Protein-based site-specific affinity reagents could allow us to antagonize harmful molecular interactions employed by pathogens, disrupt their toxic effects, and develop cutting-edge treatments. Consequently, the ability to express and purify rationally-designed proteins is the key to assessing their structure and function. To increase solubility and stability during recombinant expression, a genetic fusion to a small-ubiquitin-like-modifier (SUMO) is commonly used. The highly-specific SUMO protease is then used to cleave the SUMO fusion, leaving a completely intact and unmodified protein-of-interest. The current protease used originates from Saccharomyces cerevisiae. Known as Ulp1, the instability of this enzyme due to thermosensitivity and solvent-exposed hydrophobicity presents considerable limitations and can result in incomplete digestion of SUMO-tagged constructs. We present two approaches to mitigate these complications. First, we have discovered a homologous protease with a conserved catalytic site in the thermophilic eukaryote Chaetomium thermophilium. Second, using the Baker Lab’s pioneering computational design program, Rosetta, we have exchanged the hydrophobic surface residues of Ulp1 with hydrophilic side chains with the aim of improving stability in aqueous solution. We have characterized the kinetic function of these protease variants by monitoring digest efficacy through gel-based assays. Effectively, the improved proteases can equip the scientific community with a powerful new tool to facilitate protein-based drug design research.

Many infectious diseases are caused by bacteria growing in biofilms, a group of cells that stick together and adhere to surfaces, such as the human lung or implanted medical devices. Biofilms affect millions of people worldwide each year, often contributing to death. When a biofilm is successfully established, bacteria are up to 1,000-times more resistant to antimicrobial agents and extremely resistant to clearance by the immune system. These molecular fortifications make bacterial infections difficult to treat, with the possibility of relapse weeks or months after the use of antibiotics. Many infectious diseases caused by biofilm-forming bacteria disproportionately affect the elderly and immunocompromised, like Legionella and Bordetella, which cause pulmonary infections. My research focuses on preventing biofilm formation, and forcing bacteria that have already formed a biofilm to disperse. Molecular engineering was used to design a protein to activate LapG, a protein effector responsible for balancing whether the bacteria are free floating versus anchored. When LapG is active, bacteria shed their adhesive proteins anchoring them to surfaces, and become free floating. I am trying to develop a protein-based drug to fight biofilm-based infections by permanently switching LapG to the “on position.” Because this drug will be made of protein, there will be no toxic breakdown products, and the highly specific binding inherent to protein-based drugs will minimize the risk of side effects. Furthermore, protein-based drugs are genetically encodable, allowing us to genetically modify self-replicating probiotics, such as yogurt cultures or baker’s yeast, to produce this medicine. This will empower low cost drug manufacturing and distribution because local communities in developing countries can produce these therapeutics using readily available cooking equipment and ingredients, and administer them without fear of overdose or toxicity. The goal is to test the LapG binder in animal models of chronic infection, eventually moving to human clinical trials.

Small Ubiquitin-like Modifier, SUMO for short, is a protein involved in cellular regulation which can also be used as a biochemistry tool to express difficult-to-express proteins in the lab. I am investigating several SUMO proteases in order to learn more about the regulatory pathways it is involved in. We are focused on the traditionally used protease from Saccharomyces cerevisiae, a thermophilic version from Chaetomium thermophilium, as well as several redesigns of a homologous protein from the Saccharomyces SUMO protease to improve solubility by mutating solvent-exposed hydrophobic amino acids to polar amino acids using a computational protein design program called Rosetta. I am using thermal melting techniques to find melting points and gel comparison techniques to analyze the efficiency of each protease. Understanding of the kinetics and properties of these proteases and comparing them to each other will yield a greater understanding of how the system works, which versions are more efficient for in vitro use, and how the system can be manipulated in vivo.The goal of my research is to create a set of biochemical tools associated with SUMO proteins to be used in cancer research, as the SUMO system is known to be dysregulated in cancer. Understanding of the role SUMO plays in cancer will provide context for the development of a new generation of therapeutics, and understanding of the SUMO system as a whole will improve a current biochemical technique.

The embryonic development of vertebrates involves the anterior movement of progenitor cells, which form and proliferate at the posterior end of the embryonic body and then move anteriorly to differentiate into neural and muscle cells. Our interest is in understanding how these progenitor cells migrate, and how their movement is regulated, using the zebrafish as a model system because of its outstanding optical qualities. In my project, I am examining the role of specific proteins that are hypothesized to be important in this migration. To alter the expression of these proteins, I injected messenger RNAs encoding these proteins and also some newly developed transgenic lines where the expression of these proteins is regulated by a short period heat shock. For the mRNA injections I examined the phenotype of the whole embryo, whereas with the transgenic lines I microdissected the most posterior end of the growing embryo (called the “tailbud”) and imaged cell movement using a confocal microscope, with image analysis using software such as Slidebook and ImageJ. My results show that a change in the expression of several interesting proteins produced abnormal embryos and yielded abnormal movement of progenitor cells and impaired formation of the tail. These results provide useful information that helps us understand vertebrate development, including human embryogenesis.

Galanin is a small protein found in the gastrointestinal tract, and several places within the central nervous system. My project is to test the function of galanin-expressing neurons in the hindbrain of mice, specifically the projection from the Nucleus of the Solitary Tract to the Parabrachial Nucleus. Contrary to effects shown in other brain areas, my preliminary data suggest that the activation of galanin expressing neurons in in the hindbrain decreases feeding, in mice. These neurons are activated artificially in genetically modified mice, allowing us to then measure food intake and body weight. Their activation decreases food intake under normal conditions, but not with prior food restriction. I predict that these neurons are activated by visceral signals from afferents, or neurons that carry input from sensory organs, which are then responsible for the ensuing lack of appetite. By expressing a fluorescent protein in these neurons, I can also trace this projection to see where other connections are. Then, by examining Fos, a marker of neuronal excitation, I will investigate the activation of downstream projections. This is relevant to many current health issues, such as obesity and anorexia, as the circuits involved in hunger and appetite are not entirely understood yet.