Tag Archives: Engineering

Expanding the Genetic Code Through Simultaneous Insertion of Unnatural Amino Acids

Steven Stanley, Brigham Young University

Engineering

The genetic code has long been restricted to a set of 20 fundamental building blocks called amino acids. Recent research has expanded the genetic code through unnatural amino acids (uAA), thus adding enormous possibilities to the potential chemistries of proteins. Because nature is highly selective in the protein translation process, it has proven extremely difficult to successfully insert multiple uAAs simultaneously. The incorporation of an uAA with in vitro methods typically relies on the use of the amber stop codon as a mutated insertion site. A stop codon placed at the middle of a gene can code for either the uAA or termination, thus, protein synthesis may often terminate prematurely instead of inserting the desired uAA. This inefficiency inhibits the possibility of inserting multiple uAAs simultaneously. We propose a novel method that will allow for multiple uAAs to be inserted simultaneously. Our method involves isolating a minimal set of tRNA for in vitro protein synthesis, allowing for uAA insertion to occur at codons other than the amber stop codon. My work has focused on the production of 4 versions of uAA-tRNA synthetase, a protein that charges tRNA with the uAA. We are currently growing these 4 different proteins in bulk and testing their activity. We will test them for compatibility, confirming that they do not interfere with one another and other synthetases native to our in vitro protein synthesis system. These uAA-tRNA synthetases, in conjunction with specialized tRNA, will provide the basis to efficiently incorporate multiple uAA simultaneously. The success of this project will have many practical applications ranging from new therapeutics to new methods of medical diagnosis.

Retinal Regeneration: Implications of Müller Cell Dedifferentiation

Theo Stoddard-Bennett and Steven Christiansen, Brigham Young University

Engineering

Damage to the human retina is often irreversible and so currently there are no established treatments of diseases such as dry age related macular degeneration (AMD). Dry AMD results in a loss of sight because of cell death in the macula, a centralized part of the retina which contains a high concentration of photoreceptor cells. One possible treatment would be to limit the rate of cell death within the macula, however this is not a comprehensive solution. Rather, regeneration of the photoreceptors within the retina is necessary to restore sight. In current research, Müller glia cells, a major glial component of the retina, can potentially be used as sources for photoreceptor regeneration in order to combat dry AMD due to their homeostatic regulation of retinal injury. Directed reprogramming would occur through a five step process. The Müller glia would need to undergo de-differentiation to Müller glia-derived progenitor cells (MGPCs), proliferation of MGPCs, migration of MGPCs, neuronal differentiation, and integration in order to generate retinal neurons. Müller cells can be isolated and cultured by dissociating retinal tissue in optimal media. Here we present the dissection and dissociation of rat retinal tissue to obtain purified proliferating Müller cell cultures. Our lab has tracked and modelled the rates of proliferation and phenotypically characterized the stages of proliferation. Using immunofluorescence and PCR tests to confirm purity, we will then expect to run a series of assays to identify growth factors, Wnt signals and cytokines to test the effects of retinal extracellular matrix proteins on Müller cell de-differentiation to MGPCs. The focus of our current research is the identification of reprogramming mechanisms that may possess beneficial data leading to both unique strategies for promoting retinal regeneration in mammals and clinical applications for those living with dry AMD.

Determining the Integrity of Decellularized Porcine Kidney Scaffolds

Benjamin Buttars, Jeffrey Nielson, Spencer Baker, Jonathon Thibaudeau, Angela Nakalembe, Tim
Frost, Blake Cannon, Robert Fuller, Brinden Elton, Daniel Scott, Nafiseh Poornejad,
and Cameron Bruner, Brigham Young University

Engineering

Chronic kidney disease can be treated by organ transplantation, but the number of patients on the waiting list far exceeds the number of available donors. The patients who are fortunate enough to receive an organ need to stay on immunosuppressive medication for the remainder of their lives. Non-immunogenic tissue engineered organs could replace donor organ transplantation. We hypothesize that by seeding autologous cells on an appropriate acellular scaffold we may be able to create an inexhaustible supply of non-immunogenic organs and avoid organ rejection. In this study we have used porcine kidneys as a comparative model of human kidneys. Decellularization was used to create an intact, acellular, non-immunogenic collagenous scaffold structure. We used chelating agents and osmotic shock (alternating hypotonic, hypertonic and sodium dodecyl sulfate solutions) combined with SDS to reduce detergent exposure time by 6-fold compared to published methods using only ionic and non-ionic detergents. DNA assay results demonstrated essentially complete cell removal in all applied methods (Total DNA < 50 ng/mg). Collagen and glycosaminoglycans, which are essential for cell proliferation, differentiation, and adhesion to the scaffold, were visualized to compare the efficacy of structure integrity after decellularization. Our results showed increased integrity of the scaffold with decreasing detergent exposure time. Also, the cell adhesion and proliferation capacities of the acellular scaffolds were examined using mouse endothelial cells expressing green fluorescent protein (GFP-MS1). The resulting scaffolds demonstrated an increased capacity for cell attachment, growth and differentiation. This research demonstrates the potential for creating non-immunogenic tissue-engineered organs.

Economical Rapid Production of Therapeutic Proteins using Cell -free Protein Synthesis

Hayley Ford, Kristen Wilding and Matt Schinn, Brigham Young University

Engineering

Therapeutic proteins are specially engineered proteins used to treat many large profile diseases. Such diseases include cancer, diabetes, hepatitis B/C, hemophilia, multiple sclerosis, and anemia. The use of these proteins is specific and highly successful and the demand for these proteins in rapidly increasing. One of the largest problems with the use of therapeutic proteins is the cost of making them. The cost of producing these proteins amounts to hundreds of billions of US dollars every year. There is a growing need to find better, faster, and cheaper ways to create them. As specific therapeutic proteins are coming off patent, research labs are able to explore the processes of making these drugs that have become such a large part of the pharmaceutical industry. Here we report the use of cell-free synthesis as a more cost-effective way to produce these therapeutic proteins. Cell-free protein synthesis is faster and allows for direct manipulation and control of the protein creating environment. Cell-free synthesis can produce proteins in a matter of days as opposed to the weeks it takes to produce them in vivo. The increased manipulation and control of the environment that comes with cell-free synthesis allows improved accuracy in creating the desired proteins and is more adaptable to changes if they need to be made.

Manufacture of Hemocompatible Coronary Stents

Takami Kowalski, Warren Robison, Anton Bowden, and Brian Jensen, Brigham Young University

Engineering

Using a coronary stent to expand a blocked blood vessel as a way to treat coronary heart disease has proved effective in the past. However, there are risks, such as thrombosis, that are a natural side effect of inserting a foreign object into the body. Creating a stent out of a hemocompatible material such as carbon-infiltrated carbon nanotubes could potentially resolve these issues and also make unnecessary treatments such as dual antiplatelet therapy as a way of decreasing the risk of adverse side effects. Previous research done in this lab has shown that carbon-infiltrated carbon nanotubes can be grown in a pattern defined by photolithography on a planar surface. The present work demonstrates preliminary results from patterning a flat, flexible substrate and rolling it into a cylindrical shape before growing carbon-infiltrated carbon nanotubes as a way to fabricate cylindrical stents, fulfilling all necessary specifications for a stent with the added benefit of hemocompatibility. We also demonstrate growth on curved substrates and explore process parameters for achieving good-quality CNT forests.

Cardiac Tissue Engineering

Jordan Eatough, Jeremy Struk, Andrew Priest, Brady Vance, Brielle Woolsey, Steven Balls, Camille
Brantly, Makena Ford, Brenden Herrod, and Holly Howarth, Brigham Young University

Engineering

Ischemic heart disease is the leading killer in the world; 7.4 million people died from the disease in 2012 alone. The United States contributed over 600,000 deaths to that number.The primary treatment for the disease is heart transplantation, which has two major flaws. Our goal is to help create a solution to these two problems, namely immunorejection and scarcity of donors. We will do this by creating bioartificial hearts that could be quickly and specifically grown from each patient’s cells, thus eliminating organ rejection. We also hope to overcome the problem of thrombogenesis that often occurs with bioarticial solutions. We report an economic and effective decellularization process of porcine organs with minimum damage to the cardiac extracellular matrix (cECM). DNA and histology tests were used to verify the success of decellularization. The ECM was characterized using glycosaminoglycans (GAGs) and collagen assays, as well as scanning electron microscopy (SEM). A macrophage assay was also developed to ensure non-immunogenicity of decellularized heart tissues compared to native heart tissues. Tissue castings were made to ensure that the vasculature of the heart was not damaged in the decellularization process. Our results give promise that we can create a suitable protein scaffold for recellularization. We have begun testing for large-scale recellularization, and thrombogenicity testing of the decellularized hearts. Our goal is the creation of functional human cardiac tissue grown on the scaffold of a porcine heart. Preliminary tests have shown that the ECM created by our decellularization process is not cytotoxic, and that cell growth and proliferation has been observed.

Modeling Shale Oil Pyrolysis: Semi-empirical Approach

Dan Barfuss, Brigham Young University

Engineering

Shale oil has long been seen as a source of energy that can be incorporated into existing infrastructure. It consists of kerogen (or organic matrix) bound to inorganic rock. This kerogen can be released as an oil-like substance by heating it up to high temperatures without the presence of oxygen (i.e., pyrolysis). Due to advances in NMR (Nuclear Magnetic Resonance) we were able to make an accurate structural based model that can predict the relative tar and light gas yields[1]. We modified the Chemical Percolation Devolatilization Model (CPD) of coal to fit with the more aliphatic nature of oil shale. The CPD model describes the aromatic regions as clusters and aliphatic regions as bridges. As these bridges are broken the model releases groups of clusters that will form tar. In coal the bridge breaking gives off light gases, whereas in shale oil the bridges are much heavier and mostly form tar. We built two models that accounted for this. We also used the composition of the tar and the gas found by Fletcher et. al. [2] to predict what elements would be left and the aromaticity of the carbons. We found that throughout the reaction new aromatic regions were formed. With information from this model,- we are able to better predict the products of oil shale pyrolysis, and describe what happens chemically.

Methods for Simulating SAED and Kikuchi Diffraction Patterns in Atomistic Structures

Adam Herron, Jared Thomas, Shawn Coleman, Douglas Spearot, and Eric Homer, Brigham Young University

Engineering

For many years, x-ray diffraction and electron diffraction have served as effective means to understand and classify the molecular structure of many materials. Diffraction, as a physical phenomenon, is well known and theoretical diffraction simulation is relatively simple for perfect crystalline structures of known orientation. Prior methods of diffraction simulation, however, are insufficient to predict experimental diffraction patterns of unknown crystal structures or of crystal structures with high defect density. Recent advancements in computing capability and development of atomistic simulation software have greatly enhanced our ability to predict material properties and behaviors under various conditions. Atomistic simulation has become an extremely useful tool in the analysis of dynamic chemical and mechanical systems. It can only be truly effective, however, when it models a real-world application, can be interpreted coherently, and can accurately predict future conditions. Thus, we are developing new tools that bridge the gap between electron diffraction through real materials and simulated diffraction through atomistic simulations. We present a method of generating Kikuchi Diffraction Patterns from atomistic simulation data with no a priori knowledge of the crystal structure or crystallographic orientation. Our research was inspired by the recent work of Coleman et. al. 2013 and builds on their methods of calculating diffraction intensity at discrete locations in the reciprocal domain. We improve on their method by introducing an integration of the structure factor to ensure complete capture of diffraction intensity peaks while maintaining a relatively low density of sample points. This allows us to significantly reduce the required computation time on the analysis of atomistic simulation data. We use this diffraction data to generate simulated Kikuchi Diffraction Patterns.

In Vitro Cell-Free Synthetic Biology Techniques for Optimizing Protein Yields

Conner Earl, Brigham Young University

Engineering

The emerging field of Cell-free protein synthesis enables the efficient production of complex proteins for a number of exciting applications such as medicines that better interact with the body, vaccines, antibodies, and renewable, sustainable biocatalysts. However, progress is hampered by high costs and low yields of necessary proteins. This project is designed to improve protein yields and drive down costs by studying techniques of optimization of protein yields in Cell-Free protein synthesis. Our main area of focus is the inhibition of naturally occurring ribonucleases (RNAses) which are enzymes that degrade essential elements for protein synthesis- specifically, the mRNA used to transcribe protien. One of the techniques we intend to use for inhibition of these RNAses is by complexing the RNAse with an appropriate RNAse inhibitor protein thus limiting or eliminating its function of degrading mRNA. The aims of this research project is to: (1) Identify appropriate RNAse inhibitors (2) Design and synthesize inhibitor genes (3) Express, purify and assay RNAse inhibitors (4) Improve Cell-free protein synthesis yields utilizing RNAse inhibitors for analysis of activity and effectiveness as well as the enhancement of cell-free protein synthesis yields. Accomplishing these goals will result in more efficient systems and more accurate analysis that may lead to cheaper, more readily available vaccines and pharmaceuticals produced through Cell-free protein synthesis.

Purification of Air Using Molecular Modeling and Photocatalytic Nano-Materials

Nandini Deo, University of Utah

Engineering

Air quality in the United States has come under scrutiny in recent years. Many pollutants are trapped in the air we breathe in the form of photochemical smog. The aim of this research is to aid the breakdown of these pollutants. Peroxyacetyl Nitrate (PAN) is a predominant smog species; the research conducted aims to decompose this molecule and capture the resulting particles using the photocatalytic properties of Titanium Dioxide Nano tubes. The research conducted thus far has focused on the following questions:What molecules does the thermal decomposition of PAN produce? Is there a metal substrate to attach to TiO2 Nano-materials that aids the breakdown of PAN and its decomposition products? Can a sustainable process/device be identified to functionalize these materials? Literature research shows that PAN thermally decomposes into CO_2, NO_2, methyl nitrate, and formaldehyde. Methyl Nitrate and CO_2 may be eliminated using specific experimental conditions. Hence, it can be determined that the substrate attached to TiO2 must decompose PAN, NO_2 and formaldehyde. Using the molecular modeling programs Avogadro and MOPAC, 50 metals were optimized in relation to Formaldehyde, NO_2, and PAN. To find each metal’s reactivity to each target compound, HOMO/LUMO (Highest Occupied Molecular Orbital/Lowest Occupied Molecular Orbital) energies were calculated and used to find the common reactive metals between the target compounds: Cobalt, Silver, Iridium, and Niobium. To test whether the most complex product of the PAN decomposition (Formaldehyde) will break down, a device was created using a 3-D printer and Cobalt functionalized nanotubes. Pure formaldehyde, a blank sample (no tubes), and a sample with functionalized tubes were run through the device in the form of vapor, in front of a solar simulator. The captured vapor’s GC/MS results show an almost complete breakdown of Formaldehyde with the use of the device containing the functionalized tubes.