Browsing by Subject "Biotechnology"
Now showing 1 - 13 of 13
Results Per Page
Sort Options
Item Biotechnological Approaches for Genetic Improvement of Sorghum(2013-08-05) Urriola Simons, Jazmina ItzelSorghum (Sorghum bicolor L., Moench) is the fifth most important cereal crop in the world and represents an important source of food, feed and energy in several countries. Recently, there has been an increasing interest in sorghum cultivation worldwide, since it is relatively more drought- and heat-tolerant than other cereal crops, and it is better suited for the predicted consequences of global warming. In Africa and Asia, sorghum is primarily used as food for more than 500 million people, while in the Americas and Australia, it is used mainly as a maize-substitute in livestock feed. In the United States, sorghum is also being used in the production of ethanol. In view of its diverse utility, sorghum offers a large number of target traits that could be modified to meet the required applications. In this work, we have used different genetic engineering approaches to address two important issues in sorghum: seed quality and nitrogen use efficiency. First, we examined the temporal and spatial activity of a rice glutelin gene (GluA-2) promoter, in transgenic sorghum. Results from quantitative and histochemical GUS assays, as well as from transcript analyses, showed that this promoter is highly active during the middle stages of sorghum seed development and that it controls transgene expression specifically in the seed endosperm. This means that the GluA-2 promoter can serve as a useful tool in introducing novel traits into sorghum seed in order to improve the quality of this important cereal. Furthermore, we investigated the effects of cytosolic glutamine synthetase (GS1) and alanine aminotransferase (AlaAT) gene overexpression on nitrogen metabolism and plant growth in sorghum. T_(2) generation plants transformed with a sorghum GS1 gene (Gln1) driven by the maize ubiquitin promoter exhibited enhanced grain yield and biomass accumulation under optimal nitrogen levels.Item Comparison of glufosinate-tolerant, glyphosate-tolerant, and non-transgenic cotton weed control systems(2005-05) McCormick, Kenneth M.; Keeling, Wayne; Dotray, Peter A.; Boman, Randal K.; Segarra, Eduardo; Baughman, Todd A.Two irrigated and two dryland studies were conducted in 2003 and 2004 to compare net returns between glufosinate- and glyphosate-tolerant, and conventional cotton weed management systems. Herbicides within each weed control system were sprayed as needed based on recommended label rates and limitations, and were made independent of the weed control inputs within other systems. Weed control system costs were calculated using seed costs including technology fees, herbicide and application costs, and mechanical inputs, and lint yields were determined. The net returns above weed control system costs in 2003 with the glyphosate-tolerant system was $1,514/ha in the Lubbock irrigated study, while the glufosinate-tolerant system and conventional system were $1,131 and $889/ha, respectively. The net returns above weed control system costs were similar and ranged from $780 to $854/ha in 2004. Net returns above weed control system costs were similar for all three systems when averaged over years at the Lubbock dryland location and in 2003 at a second dryland location near Lockett. The glufosinate- and glyphosate-tolerant systems had similar net returns when averaged over years at an irrigated study near New Deal, and were greater than the conventional system. Unlike the other three trials, the conventional weed control system generated greater net returns above weed control system costs at Lockett in 2004 compared to the glyphosate-tolerant system. The glyphosate-tolerant system generally required less input to maintain effective weed control compared to the glufosinate-tolerant system, while the glufosinate-tolerant system required less input compared to the conventional system.Item Comparison of glufosinate-tolerant, glyphosate-tolerant, and non-transgenic cotton weed control systems.(Texas Tech University, 2005-05) McCormick, Kenneth M.; Keeling, Wayne; Dotray, Peter A.; Boman, Randal K.; Segarra, Eduardo; Baughman, Todd A.Two irrigated and two dryland studies were conducted in 2003 and 2004 to compare net returns between glufosinate- and glyphosate-tolerant, and conventional cotton weed management systems. Herbicides within each weed control system were sprayed as needed based on recommended label rates and limitations, and were made independent of the weed control inputs within other systems. Weed control system costs were calculated using seed costs including technology fees, herbicide and application costs, and mechanical inputs, and lint yields were determined. The net returns above weed control system costs in 2003 with the glyphosate-tolerant system was $1,514/ha in the Lubbock irrigated study, while the glufosinate-tolerant system and conventional system were $1,131 and $889/ha, respectively. The net returns above weed control system costs were similar and ranged from $780 to $854/ha in 2004. Net returns above weed control system costs were similar for all three systems when averaged over years at the Lubbock dryland location and in 2003 at a second dryland location near Lockett. The glufosinate- and glyphosate-tolerant systems had similar net returns when averaged over years at an irrigated study near New Deal, and were greater than the conventional system. Unlike the other three trials, the conventional weed control system generated greater net returns above weed control system costs at Lockett in 2004 compared to the glyphosate-tolerant system. The glyphosate-tolerant system generally required less input to maintain effective weed control compared to the glufosinate-tolerant system, while the glufosinate-tolerant system required less input compared to the conventional system.Item Consumer approval of genetic modification of food products: a comparison of United States and South Korean perspectives(Texas A&M University, 2004-09-30) Gillett, Mary CapertonGenetic modification presents the potential to advance not only agricultural production but to increase quality of life as well. The potential this innovation presents will be irrelevant if the public is unwilling to accept and adopt it. The following study examines public perceptions of biotechnology, specifically the consumer approval of genetically modified food products. This study was based on data collected from a national survey conducted in both the United States and South Korea. The United States survey was designed to be nationally representative and consisted of 1201 respondents. The South Korean survey was also designed to be nationally representative and consisted of 1054 respondents Analysis was conducted using two questions from the survey questionnaire as dependent variables: (1) approval of the use of genetic modification in the creation of plant-based food products, and (2) approval of the use of genetic modification in the creation of animal-based food products. This study utilized probit models for binary choice and ordered probit models to analyze the likelihood of consumer approval of the use of genetic modification for the creation of food products. Findings indicated that consumers in the U.S. and South Korea who possessed an accurate knowledge of the applications and outcomes of GM technology were more likely to approve of its use for the creation of foods than those who had inaccurate or no knowledge of the technology. Additionally, the majority of consumers in the U.S. and South Korea believe that GM foods should be labeled as such. Those consumers who felt GM labeling to be necessary were less likely to approve of the GM of foods than those who did not feel GM labeling to be necessary. It was also found that consumers in both countries are less approving of the GM of animals than the GM of plants. Consumer approval of the use of genetic modification in the creation of food products can be increased with proper education that provides accurate knowledge of the applications of GM. Labeling of GM products is likely to result in a decrease in demand, which may be offset by public educational campaigns.Item Decoding the antibody repertoire : high throughput sequencing of multiple transcripts from single B cells(2015-05) Dekosky, Brandon James; Georgiou, George; Ellington, Andy; Contreras, Lydia; Ehrlich, Lauren; Maynard, JenniferNext-generation (high throughput) DNA sequencing of immunoglobulin variable region and T-cell receptor gene repertoires is providing critical information for understanding adaptive immune responses and for diagnostic and therapeutic applications¹⁻⁴. However, existing immune repertoire sequencing technologies yield data on only one of the two chains of immune receptors and thus cannot provide information on the identity of immune receptor pairs encoded by individual B or T lymphocytes⁵⁻⁷. This work directly addressed these limitations by developing two new technologies for sequencing the complementary DNA (cDNA) of multiple mRNA transcripts from isolated single cells with very high throughput. In these methods, cells are sequestered into individual compartments and lysed in situ to capture single-cell mRNA onto magnetic beads, then the magnetic beads are used as template for RT-PCR reactions inside emulsion droplets that physically link cDNA of multiple transcripts for subsequent analysis by high-throughput DNA sequencing. Experimental throughput of over 2x10⁶ cells in a single day with antibody heavy and light chain pairing accuracy greater than 97% was demonstrated with in vitro expanded human B cells. These new single-cell sequencing technologies were also applied for rapid discovery of new human antibodies and for analysis of the human immune response to vaccination. Finally we applied the techniques developed here to gain new insights regarding development of the antibody repertoire through high-throughput and high-resolution examination of naïve and memory B-cell compartments in healthy human donors.Item Evaluation of Bacillus thuringiensis technology in Texas corn production(Texas Tech University, 2003-12) Youngblood, Jay LeeNot available.Item Exploring the emerging properties of novel GFP-like fluorescent proteins(2013-08) Hunt, Marguerite E; Matz, Mikhail V.In 2008 the Nobel Prize in Chemistry was awarded to the scientists who revolutionized biomedical technology by isolating, characterizing, and pioneering the use of a green fluorescent protein (GFP) from a humble hydrozoan jellyfish. Now numbering in the hundreds of colors and applications, fluorescent protein (FP) tools have facilitated the explosion of biological knowledge elucidated by a technology that can label DNA or RNA, track protein expression, and identify protein interactions. The development of the large variety of FP biotechnology available today has been due to the need for expanded color palettes and applications, and more efficient functionality. Yet, as our understanding of the biochemical and spectral characteristics of these genetically-encoded, self-assembling proteins has expanded, our comprehension of the biological function of FPs in the host organisms has remained inadequate. While the need for novel FP laboratory applications still continues, the new focus in the field of fluorescent proteins is moving to also characterize their biological functions. In this research compilation, the identification of three groups of new fluorescent proteins from marine copepods and hydrozoans has provided a collection of eleven FPs exhibiting previously uncharacterized colors, and biochemical and structural features. The green FPs from copepods are the brightest wild-type FPs identified and support the hypothesized biological function of fluorescence as counter-shading in the marine environment where these animals live. The FPs from the siphonophore and anthoathecate jelly, both hydrozoan animals, are comprised of tandemly expressed fluorescent protein units, a solution to the oligomeric structure common to most FPs that suggests a novel structure-function relationship. The fluorescent proteins from Obelia reveal a novel hydrozoan cyan FP, previously uncharacterized higher-order structural complexes, and have initiated the work to describe the biological function of these proteins as potential regenerators of their internal bioluminescent light sources. All eleven fluorescent proteins may also be adapted for FP technology.Item Group II intron mobility and its applications in biotechnology and gene therapy(2005) Karberg, Michael Steven; Lambowitz, AlanMobile group II introns use a mobility mechanism termed retrohoming in which the excised intron RNA inserts directly into a double-stranded DNA target site and is then reverse transcribed by the intron-encoded reverse transcriptase. The DNA target site for most group II introns includes ~30 nucleotides surrounding the intron insertion site, with a ~15 nucleotide region recognized by base pairing to the intron RNA. Alteration of the base-pairing interaction permits retargeting of the intron into virtually any desired DNA target. Here, I present the detailed base pairing requirements of the Ll.LtrB intron with its DNA target, including the minimal requirements for protein recognition. I developed a procedure for using retargeted group II introns for highly efficient chromosomal gene disruption in Escherichia coli and other bacteria. The retargeted introns can also be used to introduce targeted double-strand breaks, which can be repaired by cotransformation with a homologous DNA fragment, enabling the introduction of point mutations into chromosomal genes without the addition of a selectable marker. I investigate the in vivo splicing and mobility characteristics of a number of other group II introns. Those originally identified in Pylaiella littoralis and the Archaen Methanosarcina acetivorans were found to splice in vivo, while an intron identified in E. coli, and two in Novosphingobium aromaticivorans showed both splicing and mobility. I present the DNA target-site requirements of significantly mobile introns from E. coli and N. aromaticivorans, and their potential applications in biotechnology and genetic engineering are discussed.Item Mobile group II intron : host factors, directed evolution, and gene targeting in human cells(2014-05) Truong, David Minh; Lambowitz, AlanMobile group II introns are retroelements that are found in prokaryotes, archaea, and the organelles of plants and fungi, but not in the nuclear genomes of eukaryotes. They consist of a catalytically active RNA and intron-encoded reverse transcriptase, which together promote site-specific integration into DNA sites in a mechanism called retrohoming. The group II intron Ll.LtrB has been developed into a programmable, DNA-targeting agent called "targetron", which is widely used in bacteria and an attractive technology for gene targeting in eukaryotes. However, group II intron genome targeting in human cells has not been equivocally shown. This dissertation focuses on the hypothesis that the low Mg2+-concentrations found in higher eukaryotes present a natural barrier to group II introns. First, I studied E. coli host proteins that aid group II intron retrohoming and found that synthesis of a second DNA-strand relies on host replication restart proteins. Next, I demonstrated that mutations in the distal stem of the catalytic core domain V (DV) improve Ll.LtrB retrohoming in a low Mg2+-concentration E. coli mutant and in biochemical assays. These results suggest that DV is involved in an RNA-folding step that becomes rate limiting at low Mg2+. Subsequently, I performed directed evolution of the intron RNA by injecting in vitro prepared mutant intron libraries into Xenopus laevis oocyte nuclei. The mutations were analyzed using Roche 454 sequencing to generate an intron fitness landscape, which revealed conserved positions and potentially beneficial mutations, enabling enhanced retrohoming in Xenopus oocytes. Finally, I used a hybrid Pol II/T7 Ll.LtrB eukaryotic expression system to show that high exogenous MgCl2 in the growth media enables retrohoming into plasmids and genomic DNA in human cells. In vivo directed evolution and mutation analyses using PacBio RS circular consensus sequencing indicated that only a few mutations may improve intron activity in human cells. This dissertation provides evidence that efficient group II intron retrohoming in human cells is limited by low Mg2+-concentrations and develops new approaches for overcoming this limitation to enable use of group II introns for gene targeting in higher organisms.Item Next generation approaches toward engineering therapeutic proteases(2012-05) Pogson, Mark Wilson; Iverson, Brent L.; Georgiou, GeorgeEngineering protease substrate specificity and selectivity has the potential to yield entirely new possibilities in the analytical, biotechnological, and therapeutic domains. For example, therapeutic applications can be envisioned in which engineered proteases could replace antibodies by irreversibly inactivating a large excess of disease-associated target proteins in a catalytic fashion. Technological advances in molecular biology have made laboratory-based evolution techniques for protein engineering readily accessible. However, the ability to interrogate the activities and substrate preference of large numbers of protease variants is predicated on the availability of quantitative high-throughput assays that maintain the essential link between genotype and phenotype. In this work we have investigated a variety of novel single cell fluorescence assays and selections for engineering protease substrate specificity and selectivity, and demonstrated the utility of some of these systems for the engineering of novel enzymes. The second chapter of this dissertation reports the isolation of a highly active ([chemical formula]) variant of the Escherichia coli endopeptidase OmpT that selectively hydrolyzes peptides after 3-nitrotyrosine while effectively discriminating against similar peptides containing unmodified tyrosine, sulfotyrosine, phosphotyrosine and phosphoserine. The isolation of protease variants that can discriminate between substrates based on the posttranslational modification of Tyr was made possible by implementing a multi-color flow cytometric assay using multiple simultaneous counter-selection substrates for the screening of large mutant libraries. While primary sequence recognition may suffice for proteomic applications, many therapeutic applications of engineered proteases will require the cleavage of folded protein targets. Unfortunately, we have found that engineered proteases that can cleave peptides very efficiently are often unable to digest the same sequences inserted into the loop regions of a folded protein. The logical conclusion, then, is that an entire target protein or at least a protein domain, rather than peptide segments, must be incorporated into protease engineering screening assays. As a critical first step toward the development of next generation, single cell screening systems for therapeutic protease engineering we have developed novel assays that exploit cell surface capture of exogenous protein substrates. One assay (Chapter 3) relies on an autoinhibited protein fusion that capitalizes on the p53 antagonist MDM2 as a detector of protease activity in addition to its utility as a counter-selection substrate. Using this system we successfully isolated OmpT variants that selectively cleave a designed site within our autoinhibited substrate. A second high-throughput screen (Chapter 4) monitors native protein cleavage. Target proteins are captured at the cell surface using a polycationic tail, incorporating counter-selection, and the proteolytic state of the substrate can be monitored using epitope tags fused to the N-and C-termini and fluorescently labeled anti-epitope tag antibodies.Item Novel tools for engineering eukaryotic cells using a systems level approach.(2013-05) Lanza, Amanda Morgan; Alper, Hal S.; Jayaram, Makkuni; Maynard, Jennifer A; Wang, Daniel IC; Georgiou, GeorgeEngineered cellular systems are a promising avenue for production of a wide range of useful products including renewable fuels, commodity and specialty chemicals, industrial enzymes, and pharmaceuticals. Achieving this breadth of biological products is facilitated by the diversity of organisms found in nature. Using biological and engineering principles, this diversity can be harnessed to make efficient and renewable bio-based products. Such advancements rely upon our ability to modify host genetics and metabolism. This work focuses on the development of new biotechnological tools which enable cellular engineering, and the implementation of these tools in eukaryotic systems. Mammalian cell engineering has important implications in protein therapeutics and gene therapy. One major limitation, however, is the ability to predictably control gene expression. We address this challenge by examining critical aspects of gene expression in human cells. First, we evaluate the impact of selection markers, a common mammalian expression element, on cell line development. In doing so, we determine that Zeocin is the best selection agent for human cells. Next, we identify loci across the genome that support high level expression of recombinant DNA and demonstrate their advantage for stable integration. Finally, we optimize a Cre recombinase based methodology that enables efficient retargeting of genomic loci. Collectively, this work augments the current genetic toolbox for human cell lines. Beyond basic gene expression, there is interest in understanding global interactions within the cell and how they relate to phenomena including gene regulation, expression and disease states. Although our tools are not yet sufficient to study these phenomena in many hosts, methods can be developed in lower eukaryotes and then adapted for more complex hosts later. We demonstrated two methods in S. cerevisiae that utilize a systems-level approach to understand complex phenotypes. First, we developed condition-specific codon optimization that utilizes systems biology information to optimize gene sequence in a condition-specific manner. Additionally, we developed a Graded Dominant Mutant Approach which can be used to dissect multifunctional proteins, understand epigenetic factors, and quantitatively determine protein-DNA interactions. Both can be implemented in many cellular hosts and expand our ability to engineer complex phenotypes in eukaryotic cell systems.Item Studies in pharmaceutical biotechnology : protein-protein interactions and beyond(2011-05) Umeda, Aiko; Zhang, Zhiwen Jonathan; Georgiou, George; Liu, Hung-wen; Johnson, Kenneth A.; Browning, Karen S.Pharmaceutical biotechnology has been emerging as a defined, increasingly important area of science dedicated to the discovery and delivery of drugs and therapies for the treatment of various human diseases. In contrast to the advancement in pharmaceutical biotechnology, current drug discovery efforts are facing unprecedented challenges. Difficulties in identifying novel drug targets and developing effective and safe drugs are closely related to the complexity of the network of interacting human proteins. Protein-protein interactions mediate virtually all cellular processes. Therefore both identification and understanding of protein-protein interactions are essential to the process of deciphering disease mechanisms and developing treatments. Unfortunately, our current knowledge and understanding of the human interactome is largely incomplete. Most of the unknown protein-protein interactions are expected to be weak and/or transient, hence are not easily identified. These unknown or uncharacterized interactions could affect the efficacy and toxicity of drug candidates, contributing to the high rate of failure. In an attempt to facilitate the ongoing efforts in drug discovery, we describe herein a series of novel methods and their applications addressing the broad topic of protein-protein interactions. We have developed a highly efficient site-specific protein cross-linking technology mediated by the genetically incorporated non-canonical amino acid L-DOPA to facilitate the identification and characterization of weak protein-protein interactions. We also established a protocol to incorporate L-DOPA into proteins in mammalian cells to enable in vivo site-specific protein cross-kinking. We then applied the DOPA-mediated cross-linking methodology to design a protein probe which can potentially serve as a diagnostic tool or a modulator of protein-protein interactions in vivo. To deliver such engineered proteins or other bioanalytical reagents into single live cells, we established a laser-assisted cellular nano-surgery protocol which would enable detailed observations of cell-to-cell variability and communication. Finally we investigated a possible experimental scheme to genetically evolve a fluorescent peptide, which has tremendous potential as a tool in cellular imaging and dynamic observation of protein-protein interactions in vivo. We aim to contribute to the discovery and development of new drugs and eventually to the overall health of our society by adding the technology above to the array of currently available bioanalytical tools.Item The economics of plant stress reduction through biotechnology: an application to the northern plains region of Texas(Texas Tech University, 1996-12) Middleton, Marty R.Since the turn of the century, technological innovation in production agriculture has caused far-reaching changes in the techniques farmers use to produce agricultural commodities in the United States, The transition from horsepower to mechanical power, the widespread use of chemicals, and the development of new and improved seed varieties have resulted in substantial and continuing increases in agricultural productivity. Revolutions in agricultural production, such as these, have significantly increased the quantities of many agricultural commodities produced in the United States and around the world. These increased quantities have generally lead to significant shifts in total supplies of many commodities. Because of impacts on prices and availability of consumer products derived from agricultural output, shifting supplies have had meaningful social and economic impacts in society, and also in the agricultural community. Common in most American industries, widespread expectations are for technological progress to continue to play a fundamental role in the production of agricultural commodities.