Biotechnology Commons^™

Investigating The Potential Of A Cell-Based Gene Editing Therapy For Inherited Metabolic Liver Disease, Ilayda Ates

All Dissertations

Inherited metabolic diseases (IMDs) affecting the liver are relatively rare but collectively have a prevalence of 1 in 800 live births. These diseases result from autosomal recessive single-gene mutations, leading to organ dysfunction and potentially fatal consequences if left untreated. One potential therapeutic strategy for IMDs of the liver involves using CRISPR-Cas9-induced loss of function mutations. However, translating this approach into the clinic is limited by the need for safe and effective CRISPR delivery methods. Adeno-associated viral vectors (AAVs), commonly used for CRISPR delivery, are associated with significant safety and efficacy concerns, including risks for immunogenicity, off-target mutagenesis, and genotoxicity …

Laser Inscribed Graphene Aptasensor For Detection Of Apple Stem Pitting Virus, Yifan Tang

All Theses

Apple stem pitting virus (ASPV) is a latent RNA virus that could cause significant loss to apple production; early-stage field diagnosis could reduce the economic loss caused by ASPV infection. Biosensor is a promising user-friendly detection tool that could transduce biological or chemical interactions into electric signals. Among various types of biosensors, aptasensor, which use aptamers as recognition elements show potential as a high throughput, in-field test. The goal of this thesis is to develop an electrochemical aptasensor testing system for ASPV detection. The quality control study of laser inscribed graphene (LIG) based working electrode of the biosensor was conducted …

Sea-Phages Course-Based Undergraduate Research Experience For Creating A Biotechnology Workforce Development Pipeline, Daphne Fauber, Kari Clase, Carol Weaver

Graduate Industrial Research Symposium

Purdue University has been a member of the SEA-PHAGES (Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science) program since 2011. This program, created in conjunction with the Howard Hughes Medical Institute (HHMI), facilitates undergraduate curriculum for the authentic discovery of novel bacteriophages within the classroom. Since joining the program, undergraduates at Purdue have harnessed wet lab and bioinformatics principles to contribute over 200 previously uncharacterized bacteriophages and 25 novel genomes to the wider scientific literature. The SEA-PHAGES classes at Purdue have resulted in tangible professional deliverables for students through conference presentations and publications. Student outcomes also include transferable skills …

Frontiers In The Self-Assembly Of Charged Macromolecules, Khatcher O. Margossian

Doctoral Dissertations

The self-assembly of charged macromolecules forms the basis of all life on earth. From the synthesis and replication of nucleic acids, to the association of DNA to chromatin, to the targeting of RNA to various cellular compartments, to the astonishingly consistent folding of proteins, all life depends on the physics of the organization and dynamics of charged polymers. In this dissertation, I address several of the newest challenges in the assembly of these types of materials. First, I describe the exciting new physics of the complexation between polyzwitterions and polyelectrolytes. These materials open new questions and possibilities within the context …

Real-Time Monitoring Of Cell Death Progress Using Capacitance Spectroscopy, Suyang Wu

Electronic Theses and Dissertations

Biologics, including the monoclonal antibody (mAb), has experienced rapid development in the last decade. However, the price of biologics is often prohibitively high because of the low process efficiency. Delaying the inevitable cell death improves the productivity of upstream bioprocessing, whose success relies on monitoring the cell death onset that indicates the timing for preventive actions.

This study proposes to develop a real-time monitoring model that quantifies the dying cell percentage in lab-scale bioreactors using capacitance spectroscopy. The capacitance spectroscopy contains cell death-related information due to various physical properties changes during the cell death process, e.g., cytoplasmic conductivity change. The …

Toward Lignin Valorization: Development Of Rhodococcus Opacus Pd630 As A Chassis For Triacylglycerol (Tag) Production From Recalcitrant Aromatic Feedstocks, Rhiannon R. Carr

McKelvey School of Engineering Theses & Dissertations

The advent of the industrial era was precipitated by the discovery of fossil fuels, and ushered in unprecedented changes for humanity included but not limited to the development of rapid transit and communications, improvements to food distribution and preservation, the mass production of goods, and a radical rearrangement of communities from relatively small enclaves to metropolises. With all the benefits, however, come considerable costs, especially to the global environment. Greenhouse gas emissions, built up over centuries of unregulated combustion, have precipitated a rate of global temperature change unparalleled in the 4.5 billion-year history of this planet. In order to preserve …

Modeling The Effect Of Igg Subclasses And Specificity In The Translocation Of Monoclonal Antibodies Across The Placental Barrier, Sayuri Tais Miyamoto Magnabosco

ENGS 88 Honors Thesis (AB Students)

Infections are responsible for over half a million neonatal deaths every year (Lawn et al., 2014). Thus, there is huge interest in leveraging maternal immunization against infectious diseases to grant fetal protection during its development through the vertical transferring of IgG antibodies, the only Ig subclass that can significantly cross the placental barrier. Studies about vertical immunization rely on in-vitro models to extrapolate physiological conditions of the human placenta. The BeWo Transwell model (Bode et al., 2006) presents itself as a reliable model to mimic the transplacental transport mechanism of antibodies (Ellinger et al., 1999; Poulsen et al., 2009) …

Micro-Physiological Models To Mimic Mucosal Barrier Complexity Of The Human Intestine In Vitro, Abhinav Sharma

Doctoral Dissertations

The mucosal barrier in the intestine is vital to maintain selective absorption of nutrients while protecting internal tissues and maintaining symbiotic relationship with luminal microbiota. This bio-barrier consists of a cellular epithelial barrier and an acellular mucus barrier. Secreted mucus regulates barrier function via in situ biochemical and biophysical interaction with luminal content that continually evolves during digestion and absorption. Increasing evidence suggests that a mucus barrier is indispensable to maintain homeostasis in the gastrointestinal tract. However, the importance of mucus barrier is largely underrated for in vitro mucosal tissue modeling. The major gap is the lack of experimental material …

Design Of A 3d Printed Bioreactor For Bone Cancer Research, Brooklyn K. Vanderwolde, Katelyn Hillson

The Journal of Undergraduate Research

Bone cancer is an aggressive disease and has peak occurrence during physiological stimulation of growth and aging. Astronauts who undergo long-term space missions also acquire an increased risk of bone tissue degeneration and cancer. Few in-vitro models currently exist capable of reproducing the complex microenvironment of bone tissue to support multicellular activity in a three-dimensional structure. This limits the ability to understand disease progression and develop suitable treatment strategies. Therefore, developing a bone tissue model and an associated bioreactor is critical to understand the risks associated with cancer progression and improve treatment and preventions related to those risks. The goal …

Higher Tensile Forces Across Cellular Junctions And An Intact Nuclear Linc Complex Is Required For Epithelial Function And Stability, Fnu Vani Narayanan

Theses and Dissertations

Recent advances in three-dimensional (3D) cell culture systems have provided key insights into the understanding of biochemical and physiological states of native tissue. A significant progress in the field of mechanobiology involves measuring cellular traction forces in a more native 3D environment. However, the effects of mechanical forces exerted across cellular junctions and the nuclear LINC complex, in an organized 3D system has not been investigated thus far. Epithelial cells spontaneously form acini (also known as cysts or spheroids) with a single, fluid-filled central lumen, when grown in 3D matrices. The size of the lumen is dependent on apical secretion …

Maximizing And Modeling Malonyl-Coa Production In Escherichia Coli, Tatiana Thompson Silveira Mello

LSU Master's Theses

In E. coli, fatty acid synthesis is catalyzed by the enzyme acetyl-CoA carboxylase (ACC), which converts acetyl-CoA into malonyl-CoA. Malonyl-CoA is a major building block for numerous of bioproducts. Multiple parameters regulate the homeostatic cellular concentration of malonyl-CoA, keeping it at a very low level. Understanding how these parameters affect the bacterial production of malonyl-CoA is fundamental to maximizing it and its bioproducts. To this end, competing pathways consuming malonyl-CoA can be eliminated, and optimal nutritional and environmental conditions can be provided to the fermentation broth. Most previous studies utilized genetic modifications, expensive consumables, and high-cost quantification methods, making …

Microfluidic Electrical Impedance Spectroscopy, John J. Foley

Master's Theses

The goal of this study is to design and manufacture a microfluidic device capable of measuring changes in impedance valuesof microfluidic cell cultures. Tocharacterize this, an interdigitated array of electrodes was patterned over glass, where it was then bonded to a series of fluidic networks created in PDMS via soft lithography. The device measured ethanol impedance initially to show that values remain consistent over time. Impedance values of water and 1% wt. saltwater were compared to show that the device is able to detect changes in impedance, with up to a 60% reduction in electrical impedance in saltwater. Cells were …

Cost-Effective Paper-Based Diagnostic Using Split Proteins To Detect Yeast Infections, Zachary R. Berglund, Kevin V. Solomon, Mohit S. Verma, Moiz Rasheed, Zachary Hartley, Kevin Fitzgerald, Kok Zhi Lee, Janice Chan, Julianne Dejoie, Makayla Schacht, Alex Zavala

The Summer Undergraduate Research Fellowship (SURF) Symposium

The common yeast infection, vulvovaginal candidiasis, affects three out of four women throughout their lifetime and can be spread to their child in the form of oral candidiasis (thrush). This disease is caused by the fungal pathogen Candida albicans, which is also a major cause of systemic candidiasis, a rarer but deadly disease with up to a 49% lethality rate. Current widely-used diagnostic methods include cell cultures, pH tests, and antibody detection, to assist effective treatment. Despite availability of various diagnostic methods, there is no inexpensive, rapid, and accurate way to detect C. albicans infection. This project aims to …

Developing Droplet Based 3d Cell Culture Methods To Enable Investigations Of The Chemical Tumor Microenvironment, Jacqueline A. De Lora

Biomedical Sciences ETDs

Adaptation of cancer cells to changes in the biochemical microenvironment in an expanding tumor mass is a crucial aspect of malignant progression, tumor metabolism, and drug efficacy. In vitro, it is challenging to mimic the evolution of biochemical gradients and the cellular heterogeneity that characterizes cancer tissues found in vivo. It is well accepted that more realistic and controllable in vitro 3D model systems are required to improve the overall cancer research paradigm and thus improve on the translation of results, but multidisciplinary approaches are needed for these advances. This work develops such approaches and demonstrates that new droplet-based cell-encapsulation …

Optimizing Genetic Manipulation Of Methanogens Through Faster Cloning Techniques, Merrisa Jennings

Biological and Agricultural Engineering Undergraduate Honors Theses

Methanogenesis is the biological production of methane. Only anaerobic archaea known as methanogens are capable of such a metabolic feat. They have strict living conditions and substrate sources which determine their rate of metabolism. This is of particular importance from a greenhouse gas reduction perspective or biogas capturing perspective. One of the best ways to optimize methanogen methane production is via genetic manipulation. The current procedures are timely though, therefore a faster cloning processes should be developed. The objective of this study was to optimize a premade genetic transformation kit known as the Gibson Kit. The Gibson Kit was supposed …

Mutagenesis Of Human Alpha-Galactosidase A For The Treatment Of Fabry Disease, Erin Stokes

Dissertations, Theses, and Capstone Projects

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of the enzyme, α-galactosidase A, which results in the accumulation of the lipid substrate. This accumulation results in obstruction of blood flow in patients and early demise at approximately 40-60 years of age. There is currently only one FDA approved treatment (Fabrazyme) classified as an enzyme replacement therapy. However, approximately 88% of patients experience a severe immune response that, rarely, can be fatal and is a huge cost burden at average $250,000 a year per patient. The structure of α-galactosidase A has been previously determined to be a …

The Response Of Schwann Cells To Weak Dc Electric Fields, Alexander T. Lai, Jianming Li

The Summer Undergraduate Research Fellowship (SURF) Symposium

Schwann cells are glial cells that serve the vital role of supporting neurons in the peripheral nervous system. While their primary function is to provide insulation (myelin) for axons, they also help regenerate injured axons by digesting severed axons and providing scaffolding to guide the regeneration process. This specific role of Schwann cells makes them highly important cellular targets following nerve injury. Although some efforts have been made to encourage Schwann cell migration after nerve damage, the use of electric fields to control cell responses remain unexplored; therefore, this experiment serves to characterize the behavior of Schwann cells to weak …

Establishing A Lung Model For Evaluation Of Engineered Lung Microbiome Therapies, Kathryn F. Atherton, Stephen Miloro, Jenna Rickus

The Summer Undergraduate Research Fellowship (SURF) Symposium

Benzene, a toxin and carcinogen found in air polluted by cigarette smoke, car exhaust, and industrial processes, is associated with the development of leukemia and lymphoma. Other than avoiding exposure, there is no current method to deter the effects of benzene. One potential strategy to prevent these effects is to engineer the bacteria of the human lung microbiome to degrade benzene. To evaluate this novel approach, we must verify that the bacteria remain viable within the lung microenvironment. To do so, lungs were harvested from rats and swabbed to determine the contents of the original lung microbiome. Then green fluorescent …

Managing Exoelectrogenic Microbial Community Development Through Bioprocess Control For Conversion Of Biomass-Derived Streams, Alex James Lewis

Doctoral Dissertations

Bioelectrochemical systems are an emerging technology capable of utilizing aqueous waste streams generated during biomass conversion of lignocellulosic feedstocks to produce valuable co-products and thus, have potential to be integrated into biorefineries. In a microbial electrolysis cell, organic compounds are converted to electrons, protons, and CO₂ by fermentative and exoelectrogenic bacteria in the anode compartment. By having the ability to extract electrons from waste streams, these systems can treat water while also producing hydrogen, and thus can improve the efficiency of biomass to fuel production by minimizing external hydrogen requirement and enabling water recycle. The overall goal of this …

Fractionation And Characterization Of Lignin Streams From Genetically Engineered Switchgrass, Enshi Liu

Theses and Dissertations--Biosystems and Agricultural Engineering

Development of biomass feedstocks with desirable traits for cost-effective conversion is one of the main focus areas in biofuels research. As suggested by techno-economic analyses, the success of a lignocellulose-based biorefinery largely relies on the utilization of lignin to generate value-added products, i.e. fuels and chemicals. The fate of lignin and its structural/compositional changes during pretreatment have received increasing attention; however, the effect of genetic modification on the fractionation, depolymerization and catalytic upgrading of lignin from genetically engineered plants is not well understood. This study aims to fractionate and characterize the lignin streams from a wild-type and two genetically engineered …

Examination Of Pseudomonas Fluorescence As A Recombinant Expression Host: Cloning, Expression, And Chromatography, Ahmed K.Ali Elmasheiti

Graduate Theses and Dissertations

In an effort to expand the pool of bacterium useful for biotechnology applications, Pseudomonas fluorescens, a common gram negative microbe, was examined for its ability to function in a recombinant setting. P. fluorescens is ubiquitous in nature and was initially identified as a soil bacterium found in dirt and is typically associated with plant material. Past literature indicates that it shared characteristics common to Escherichia coli and Bacillus subtilis, including simple growth conditions and potential cloning vectors, providing motivation to look into both the upstream and downstream characteristics of this bacterium. First, it was demonstrated that P. fluorescens could be …

Development Of In Vivo Systems For Detecting And Studying Ribosome Inhibition By Small Molecules, Shijie Huang

Chemistry and Chemical Biology ETDs

The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E. coli strains harboring an orthogonal ribosome controlled green fluorescent protein reporter that produce fluorescent signal when the O-ribosome is inhibited. As a proof …

Fermentative Processes Requiring Low Solubility Feed Gases: An Investigation Into Gas-Dependent Microorganisms, Eric W. Doerr

Electronic Thesis and Dissertation Repository

Two bioprocesses were separately investigated based on their common interest of using gaseous substrates that have low solubility. The first process involved the development of a mixed culture using two organisms capable of utilizing glycerol and carbon monoxide separately to increase biobutanol production, while the second process involved an investigation of different production media used in aerobic xanthan production of Xanthomonas campestris with pressurization effects. It was determined that Clostridium pasteurianum should be used with an organism like Clostridium carboxidivorans or Eubacterium limosum in order for butyrate uptake at 3 g/L to occur with a minimum 0.1 g/L butyrate production. …

The Role Of E-Cadherin Force In The Maintenance Of Homeostasis In Epithelial Acini, Fnu Vani Narayanan

Theses and Dissertations

Numerous three-dimensional model systems have emerged for emulating the biochemical and physiological states of native tissue. Yet little is known about the effects of mechanical forces on cell behavior in the context of an organized tissue structure in three-dimensional cell-culture. Epithelial cells cultured in a three-dimensional environment comprised of extracellular matrix proteins form spheroids of polarized cells. Cellular responses to mechanical cues, generated from dynamic interactions with the extracellular matrix and neighboring cells, are known to influence cellular behavior to a great extent. Previous studies have shown that tumorigenic progression has been frequently linked to the down regulation of E-cadherin, …

Label-Free Surface-Enhanced Raman Spectroscopy-Linked Immunosensor Assay (Slisa) For Environmental Surveillance, Vinay Bhardwaj

FIU Electronic Theses and Dissertations

The contamination of the environment, accidental or intentional, in particular with chemical toxins such as industrial chemicals and chemical warfare agents has increased public fear. There is a critical requirement for the continuous detection of toxins present at very low levels in the environment. Indeed, some ultra-sensitive analytical techniques already exist, for example chromatography and mass spectroscopy, which are approved by the US Environmental Protection Agency for the detection of toxins. However, these techniques are limited to the detection of known toxins. Cellular expression of genomic and proteomic biomarkers in response to toxins allows monitoring of known as well as …

Development Of A Novel Enzymatic Pre-Treatment For Lignocellulosic Biomass, Melissa Robins, Jenna Rickus

The Summer Undergraduate Research Fellowship (SURF) Symposium

Biofuels, fuels derived directly from living matter, present a renewable and environmentally friendly alternative to petroleum based fuels. Bioethanol produced from low input energy crops or agricultural waste is a promising fuel source because it does not interfere with the human food supply chain and the ethanol produced can be blended with gasoline. These potential sources of bioethanol are not yet commercially viable due to a polymer called lignin present in the plant’s cell wall which impedes the conversion of cellulose to glucose and the eventual fermentation of glucose to ethanol. Developing new methods for the pretreatment of lignocellulosic biomass …

A Novel Synthetic Yeast For Enzymatic Biodigester Pretreatment, Tianyu Tan, Mark S. Aronson, Arren Liu, Jill H. Osterhus, Melissa Robins, Suraj Mohan, Erich Leazer, Bowman Clark, Alexa Petrucciani, Katherine Lowery, James Welch, Casey Martin, Helena Lysandrou, Michael E. Scharf, Jenna Rickus

The Summer Undergraduate Research Fellowship (SURF) Symposium

Lignin, a complex organic polymer, is a major roadblock to the efficiency of biofuel conversion as it both physically blocks carbohydrate substrates and poisons biomass degrading enzymes, even if broken down to monomer units. A pretreatment process is often applied to separate the lignin from biomass prior to biofuel conversion. However, contemporary methods of pretreatment require large amounts of energy, which may be economically uncompelling or unfeasible. Taking inspiration from several genes that have been isolated from termites and fungi which translate to enzymes that degrade lignin, we want to establish a novel “enzymatic pretreatment” system where microbes secrete these …

Selection Methods For Genetically-Modified T Cells: In Support Of Translational Therapy, David Rushworth

Dissertations & Theses (Open Access)

T cells are blood cells which organize the immune system of the host. These cells are necessary for the host to respond appropriately to threats from foreign organisms and cancerous growth. However, in the case of certain infections and cancer, T cells are unable to respond appropriately to a threat and establish immunity. This leads to disease when the infection or cancer is not sufficiently eliminated. On the other hand, T cells can lack tolerance for healthy tissue and perceive healthy tissue as infected. The ensuing over-reactive immune response also leads to disease. A delicate balance must exist between immunity …

Ex Vivo Dna Cloning, Adam B. Fisher

Theses and Dissertations

Genetic engineering of microbes has developed rapidly along with our ability to synthesize DNA de novo. Yet, even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. While technological advances have resulted in powerful techniques for in vitro and in vivo assembly of DNA, each suffers inherent disadvantages. Here, an ex vivo DNA cloning suite using crude cellular lysates derived from E. coli is demonstrated to amplify and assemble DNA containing small sequence homologies. Further, the advantages of an ex vivo approach are …

Movement Effects On The Flow Physics And Nutrient Delivery In Engineered Valvular Tissues, Manuel Salinas

FIU Electronic Theses and Dissertations

Mechanical conditioning has been shown to promote tissue formation in a wide variety of tissue engineering efforts. However the underlying mechanisms by which external mechanical stimuli regulate cells and tissues are not known. This is particularly relevant in the area of heart valve tissue engineering (HVTE) owing to the intense hemodynamic environments that surround native valves. Some studies suggest that oscillatory shear stress (OSS) caused by steady flow and scaffold flexure play a critical role in engineered tissue formation derived from bone marrow derived stem cells (BMSCs). In addition, scaffold flexure may enhance nutrient (e.g. oxygen, glucose) transport. In this …