Biological Engineering Commons^™

High-Throughput Robotic Ethanol Inhibition Assays For Engineered Thermophilic Biofuel Strains, Kevin He, Daniel Olson, Marybeth Maloney, Anthony Lanahan

Wetterhahn Science Symposium Posters

Ethanol stress assays are commonly used to evaluate microbial tolerance, metabolic adaptation, and fermentation performance. However, manual liquid handling introduces variability across replicate wells and small-volume pipetting steps, limiting reproducibility and throughput. This study developed an automated OT-2 robotic workflow to generate replicated ethanol concentration gradients for high-throughput inhibition assays in engineered thermophilic biofuel strains. Kinetic plate-reader measurements were used to quantify ethanol-dependent growth responses under anaerobic fermentation conditions. The reasearch question is: How do engineered thermophilic biofuel strains differ in ethanol-dependent growth inhibition under anaerobic fermentation conditions, and can automated robotic assays improve the reproducibility of these measurements? Can …

Metabolite Analysis To Understand Factors Limiting Microbial Fermentation At High Substrate Concentrations, Bishal D. Sharma

Dartmouth College Ph.D Dissertations

Clostridium thermocellum, a thermophilic anaerobic bacterium, holds promise as a biocatalyst for conversion of lignocellulosic biomass into biofuels. This microbe has been subjected to various metabolic engineering strategies, including the deletion of genes encoding secondary fermentation products such as H₂, formate, acetate, and lactate, as well as the expression of heterologous genes to redirect carbon flux toward ethanol production. Despite these efforts, achieving economically viable ethanol titers remains a challenge. We hypothesized that studying how intracellular metabolite concentrations change as fermentation stops could reveal metabolic bottlenecks or regulatory mechanisms limiting ethanol production at high titers. However, metabolomics studies at …

The Bacteriostatic, Regenerative, And Immunomodulatory Properties Of Extracellular Matrix Particles For Lung Injury, Keera P. Rhoads

Theses and Dissertations

Acute respiratory distress syndrome (ARDS) is a prevalent, life-threatening lung condition, affecting nearly 200,000 Americans annually, with a 40% international mortality rate. There is no cure for ARDS, and current pharmacological treatments have limited effectiveness. Symptoms can be mitigated with mechanical ventilation, though this often leads to ventilator-induced lung injuries (VILI) and puts critically ill patients at risk of infections, including ventilator-associated pneumonia (VAP). A promising therapeutic is the extracellular matrix (ECM), a complex network of structural proteins and bioactive molecules that has been shown to have anti-inflammatory properties and prevent fibrosis. We aim to utilize the regenerative and immunomodulatory …

Evaluation Of The Dnazyme Gr5 For The Determination Of Lead Speciation In Natural Waters, Gaganprit Gill

Theses and Dissertations (Comprehensive)

This thesis explores the development and application of a DNAzyme-based biosensor designed to detect labile metal species in environmental samples. Real time and on-site monitoring of labile metal fractions would make a valuable contribution to environmental management, as these fractions are the most bioavailable and pose significant toxicity risks to aquatic organisms. Conventional methods for detecting labile metals, while effective, are often burdened by high costs, complexity, and lengthy processing times, making them less ideal for rapid and widespread environmental assessments.

The first manuscript of this thesis (chapter 2) establishes the fundamental capabilities of the Pb²⁺-specific DNAzyme GR5 …

Air-Jet Bioprinting Of Stem Cell-Encapsulated Alginate Micro-Droplets For Organoid Biomanufacturing, Mirabella B. Stump

Electronic Theses and Dissertations

Biomanufactured 3D-organoids show promise for modeling and treating various conditions, particularly insulin-dependent diabetes mellitus (type-1 diabetes). Pancreatic islet organoids serve as sustainable sources of insulin-producing β-cells, potentially restoring insulin regulation in patients and providing a long-term solution for diabetes. Hydrogel droplet printing is an emerging technique in regenerative medicine due to its advantageous tunability and biocompatibility; however, challenges of consistency, quality control, and scalability persist. To address these issues, we developed a novel air-jetting based droplet bioprinting system for the generation of monodispersed alginate micro-droplets. The objective of this project was to incorporate human induced pluripotent stem cells (hiPSCs) into …

Fiber-Based Time-Correlated Single Photon Counting As A Method For Investigating Metabolism During Tetanic Contraction Of Skeletal Muscle, Kathryn Miller Priest

Graduate Theses and Dissertations

Nicotinamide adenine dinucleotide (NAD) is a cofactor that serves to shuttle electrons during metabolic processes such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) [1-5]. The reduced form of NAD, NADH, is autofluorescent, making it a measurable characteristic in the investigation of metabolic changes. The fluorescence lifetime of NADH is dependent on the state of protein binding, specifically free or bound NADH, allowing for detection of metabolic shifts between glycolysis (free NADH) and OXPHOS (bound NADH) [6-16]. Fiber-coupled time-correlated single photon counting (TCSPC) equipped with an implantable needle probe can be used to measure NADH lifetime in vivo …

Project Alien, Rebecca Mccallin, Madelyn Hoying, Alex Evans, Matthew Nestler, Karli Rae Sutton, Garett Craig, Lucia Secaia Del Cid, Alexander Guy, Rachel Fernandez, Amanda Trusiak, Paige Aley, Ingabire Gakwerere, Nina Dorfner, Maria Mosbacher, Mary Flavin, Selvin Hernandez, Audrey Steen, Benjamin Kazimer

Undergraduate Research and Scholarship Symposium

Project ALIEN is a comprehensive plan to send humans to Mars to look for life on the Martian surface while exploring the viability and adaptability of terrestrial microbes in Martian atmospheric conditions. ALIEN will use a ballistic capture trajectory to get to Mars and stay in aerostationary orbit for a 30-day surface mission, during which two surface crewmembers will perform a variety of experiments to achieve the mission’s goals of Martian microbial discovery within brines of the Gale Crater and terrestrial microbe adaptability and viability to Martian conditions. Experimentation is based on the presumption that Martian microbes are metabolically similar …

Development Of Light Actuated Chemical Delivery Platform On A 2-D Array Of Micropore Structure, Hojjat Rostami Azmand, Hojjat Rostami Azmand

Dissertations and Theses

Localized chemical delivery plays an essential role in the fundamental information transfers within biological systems. Thus, the ability to mimic the natural chemical signal modulation would provide significant contributions to understand the functional signaling pathway of biological cells and develop new prosthetic devices for neurological disorders. In this paper, we demonstrate a light-controlled hydrogel platform that can be used for localized chemical delivery in a high spatial resolution. By utilizing the photothermal behavior of graphene-hydrogel composites confined within micron-sized fluidic channels, patterned light illumination creates the parallel and independent actuation of chemical release in a group of fluidic ports. The …

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 …

Advanced Selection Methodologies For Dnazymes In Sensing And Healthcare Applications, Sandeep Kumar, Shikha Jain, Neeraj Dilbaghi, Amrik Singh Ahluwalia, Ashraf Aly Hassan, Ki-Hyun Kim

Department of Civil and Environmental Engineering: Faculty Publications

DNAzymes have been widely explored owing to their excellent catalytic activity in a broad range of applications, notably in sensing and biomedical devices. These newly discovered applications have built high hopes for designing novel catalytic DNAzymes. However, the selection of efficient DNAzymes is a challenging process but one that is of crucial importance. Initially, systemic evolution of ligands by exponential enrichment (SELEX) was a labor-intensive and time-consuming process, but recent advances have accelerated the automated generation of DNAzyme molecules. This review summarizes recent advances in SELEX that improve the affinity and specificity of DNAzymes. The thriving generation of new DNAzymes …

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 …

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 …

Enhanced Hot Electron Lifetimes In Quantum Wells With Inhibited Phonon Coupling, Hamidreza Esmaielpour, Vincent R. Whiteside, Herath P. Piyathilaka, Sangeetha Vijeyaragunathan, Bin Wang, Echo Adcock-Smith, Kenneth P. Roberts, Tetsuya D. Mishima, Michael B. Santos, Alan D. Bristow, Ian R. Sellers

Faculty & Staff Scholarship

Hot electrons established by the absorption of high-energy photons typically thermalize on a picosecond time scale in a semiconductor, dissipating energy via various phonon-mediated relaxation pathways. Here it is shown that a strong hot carrier distribution can be produced using a type-II quantum well structure. In such systems it is shown that the dominant hot carrier thermalization process is limited by the radiative recombination lifetime of electrons with reduced wavefunction overlap with holes. It is proposed that the subsequent reabsorption of acoustic and optical phonons is facilitated by a mismatch in phonon dispersions at the InAs-AlAsSb interface and serves to …

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 …

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 …

Nanobubbles Provide Theranostic Relief To Cancer Hypoxia, Christopher M. Long, Pushpak N. Bhandari, Joseph Irudayaraj

The Summer Undergraduate Research Fellowship (SURF) Symposium

Hypoxia is a common motif among tumors, contributing to metastasis, angiogenesis, cellular epigenetic abnormality, and resistance to cancer therapy. Hypoxia also plays a pivotal role in oncological studies, where it can be used as a principal target for new anti-cancer therapeutic methods. Oxygen nanobubbles were designed in an effort to target the hypoxic tumor regions, thus interrupting the hypoxia-inducible factor-1α (HIF-1α) regulatory pathway and inhibiting tumor progression. At less than 100nm, oxygen nanobubbles act as a vehicle for site-specific oxygen delivery, while also serving as an ultrasound contrast agent for advanced imaging purposes. Through in vitro and in vivo studies, …

Role Of Sumoylation In Mitochondrial Division In Tetrahymena Thermophila, Ramya Modi, James Forney

The Summer Undergraduate Research Fellowship (SURF) Symposium

SUMOylation is a post translation modification that involves the addition of a small protein called SUMO, Small Ubiquitin-like MOdifier to a target protein. It is an important mechanism for the regulation of gene expression, the maintenance of genomic stability and in modifying nuclear proteins. More recently evidence has emerged for its importance in regulating mitochondrial fission and fusion in mammalian cells. This study evaluates the parameters for optimal staining of Tetrahymena thermophila mitochondria using two different dyes and then examines different cell lines with defects in the SUMOylation pathway. The first staining method uses Mitotracker Green, a vital stain that …

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 and 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 …

Systematic Study Of The Biological Effects Of Nitric Oxide (No) Using Innovative No Measurement And Delivery Systems, Weilue He

Dissertations, Master's Theses and Master's Reports

Nitric oxide (NO) is recognized as the most important small signaling molecule in the human body. An imbalance of NO is closely associated with many serious diseases such as neurological disorders, cardiovascular diseases, chronic inflammations and cancers. Herein two chemiluminescence-based devices (a real-time NO measurement device and a controllable NO delivery device) were developed to facilitate the NO quantitative study and obtain information for NO related drug design.

The first device used for real-time measuring NO(g) flux from living cells was developed and validated. The principle was to use a two-chamber design, with a cell culture chamber and a gaseous …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

University Scholar Projects

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

Honors Scholar Theses

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Mutant Alcohol Dehydrogenase Leads To Improved Ethanol Tolerance In Clostridium Thermocellum, Steven D. Brown, Adam M. Guss, Tatiana V. Karpinets, Jerry M. Parks

Dartmouth Scholarship

Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. …

Forward Osmosis Bag: Ground Testing The Prospect Of Using Alternative Water Sources For Drinking Water, Michaela Marie Brant, Ariella Yendler, Mona Hammoudeh, Michael T. Flynn

STAR Program Research Presentations

On the STS-135 Space Shuttle mission, to be launched July 8, 2011, a forward osmosis bag (FOB) study will be conducted. At NASA Ames this summer, the ground truth testing is being conducted for results comparisons. The FOB technology is derived from a commercial product, the X-Pack water filter. Forward osmosis operates by utilizing an established concentration gradient across a semi-permeable membrane to move water molecules from one side of the membrane to the other. This concept is exploited to harvest drinking water from grey water sources such as urine, sea water, or vehicle water. In this experiment, potassium chloride …