Matching Items (534)
Filtering by
- All Subjects: Creative Project
Description
The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules with the particular property. DNA and RNA sequences with versatile functions have been identified by in vitro selection experiments, but many basic questions remain to be answered about how these molecules achieve their functions. This dissertation first focuses on addressing a fundamental question regarding the molecular recognition properties of in vitro selected DNA sequences, namely whether negatively charged DNA sequences can be evolved to bind alkaline proteins with high specificity. We showed that DNA binders could be made, through carefully designed stringent in vitro selection, to discriminate different alkaline proteins. The focus of this dissertation is then shifted to in vitro evolution of an artificial genetic polymer called threose nucleic acid (TNA). TNA has been considered a potential RNA progenitor during early evolution of life on Earth. However, further experimental evidence to support TNA as a primordial genetic material is lacking. In this dissertation we demonstrated the capacity of TNA to form stable tertiary structure with specific ligand binding property, which suggests a possible role of TNA as a pre-RNA genetic polymer. Additionally, we discussed the challenges in in vitro evolution for TNA enzymes and developed the necessary methodology for future TNA enzyme evolution.
ContributorsYu, Hanyang (Author) / Chaput, John C (Thesis advisor) / Chen, Julian (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
The production of monomer compounds for synthesizing plastics has to date been largely restricted to the petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economic feasibility. Cyanobacteria have, however, become attractive microbial factories to produce renewable fuels and chemicals directly from sunlight and CO2. To explore the feasibility of photosynthetic production of (S)- and (R)-3-hydroxybutyrate (3HB), building-block monomers for synthesizing the biodegradable plastics polyhydroxyalkanoates and precursors to fine chemicals, synthetic metabolic pathways have been constructed, characterized and optimized in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803). Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing PHB biosynthesis pathway further promoted the 3HB production. Analysis of the intracellular acetyl-CoA and anion concentrations in the culture media indicated that the phosphate consumption during the photoautotrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. Fine-tuning of the gene expression levels via strategies, including tuning gene copy numbers, promoter engineering and ribosome binding site optimization, proved critical to mitigating metabolic bottlenecks and thus improving the 3HB production. One of the engineered Synechocystis strains, namely R168, was able to produce (R)-3HB to a cumulative titer of ~1600 mg/L, with a peak daily productivity of ~200 mg/L, using light and CO2 as the sole energy and carbon sources, respectively. Additionally, in order to establish a high-efficiency transformation protocol in cyanobacterium Synechocystis 6803, methyltransferase-encoding genes were cloned and expressed to pre-methylate the exogenous DNA before Synechocystis transformation. Eventually, the transformation efficiency was increased by two orders of magnitude in Synechocystis. This research has demonstrated the use of cyanobacteria as cell factories to produce 3HB directly from light and CO2, and developed new synthetic biology tools for cyanobacteria.
ContributorsWang, Bo (Author) / Meldrum, Deirdre R (Thesis advisor) / Zhang, Weiwen (Committee member) / Sandrin, Todd R. (Committee member) / Nielsen, David R (Committee member) / Arizona State University (Publisher)
Created2014
Description
Synthetic biology is constantly evolving as new ideas are incorporated into this increasingly flexible field. It incorporates the engineering of life with standard genetic parts and methods; new organisms with new genomes; expansion of life to include new components, capabilities, and chemistries; and even completely synthetic organisms that mimic life while being composed of non-living matter. We have introduced a new paradigm of synthetic biology that melds the methods of in vitro evolution with the goals and philosophy of synthetic biology. The Family B proteins represent the first de novo evolved natively folded proteins to be developed with increasingly powerful tools of molecular evolution. These proteins are folded and functional, composed of the 20 canonical amino acids, and in many ways resemble natural proteins. However, their evolutionary history is quite different from natural proteins, as it did not involve a cellular environment. In this study, we examine the properties of DX, one of the Family B proteins that have been evolutionarily optimized for folding stability. Described in chapter 2 is an investigation into the primitive catalytic properties of DX, which seems to have evolved a serendipitous ATPase activity in addition to its selected ATP binding activity. In chapters 3 and 4 we express the DX gene in E. coli cells and observe massive changes in cell morphology, biochemistry, and life cycle. Exposure to DX activates several defense systems in E. coli, including filamentation, cytoplasmic segregation, and reversion to a viable but non-culturable state. We examined these phenotypes in detail and present a model that accounts for how DX causes such a rearrangement of the cell.
ContributorsStomel, Joshua (Author) / Chaput, John C (Thesis advisor) / Korch, Shaleen (Committee member) / Roberson, Robert (Committee member) / Ghirlanda, Gionvanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
A major goal of synthetic biology is to recapitulate emergent properties of life. Despite a significant body of work, a longstanding question that remains to be answered is how such a complex system arose? In this dissertation, synthetic nucleic acid molecules with alternative sugar-phosphate backbones were investigated as potential ancestors of DNA and RNA. Threose nucleic acid (TNA) is capable of forming stable helical structures with complementary strands of itself and RNA. This provides a plausible mechanism for genetic information transfer between TNA and RNA. Therefore TNA has been proposed as a potential RNA progenitor. Using molecular evolution, functional sequences were isolated from a pool of random TNA molecules. This implicates a possible chemical framework capable of crosstalk between TNA and RNA. Further, this shows that heredity and evolution are not limited to the natural genetic system based on ribofuranosyl nucleic acids. Another alternative genetic system, glycerol nucleic acid (GNA) undergoes intrasystem pairing with superior thermalstability compared to that of DNA. Inspired by this property, I demonstrated a minimal nanostructure composed of both left- and right-handed mirro image GNA. This work suggested that GNA could be useful as promising orthogonal material in structural DNA nanotechnology.
ContributorsZhang, Su (Author) / Chaut, John C (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2011
Description
Advances in chemical synthesis have enabled new lines of research with unnatural genetic polymers whose modified bases or sugar-phosphate backbones have potential therapeutic and biotechnological applications. Maximizing the potential of these synthetic genetic systems requires inventing new molecular biology tools that can both generate and faithfully replicate unnatural polymers of significant length. Threose nucleic acid (TNA) has received significant attention as a complete replication system has been developed by engineering natural polymerases to broaden their substrate specificity. The system, however, suffers from a high mutational load reducing its utility. This thesis will cover the development of two new polymerases capable of transcribing and reverse transcribing TNA polymers with high efficiency and fidelity. The polymerases are identified using a new strategy wherein gain-of-function mutations are sampled in homologous protein architectures leading to subtle optimization of protein function. The new replication system has a fidelity that supports the propagation of genetic information enabling in vitro selection of functional TNA molecules. TNA aptamers to human alpha-thrombin are identified and demonstrated to have superior stability compared to DNA and RNA in biologically relevant conditions. This is the first demonstration that functional TNA molecules have potential in biotechnology and molecular medicine.
ContributorsDunn, Matthew Ryan (Author) / Chaput, John C (Thesis advisor) / LaBaer, Joshua (Committee member) / Lake, Douglas (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2015
Description
Recombinases are powerful tools for genome engineering and synthetic biology, however recombinases are limited by a lack of user-programmability and often require complex directed-evolution experiments to retarget specificity. Conversely, CRISPR systems have extreme versatility yet can induce off-target mutations and karyotypic destabilization. To address these constraints we developed an RNA-guided recombinase protein by fusing a hyperactive mutant resolvase from transposon TN3 to catalytically inactive Cas9. We validated recombinase-Cas9 (rCas9) function in model eukaryote Saccharomyces cerevisiae using a chromosomally integrated fluorescent reporter. Moreover, we demonstrated cooperative targeting by CRISPR RNAs at spacings of 22 or 40bps is necessary for directing recombination. Using PCR and Sanger sequencing, we confirmed rCas9 targets DNA recombination. With further development we envision rCas9 becoming useful in the development of RNA-programmed genetic circuitry as well as high-specificity genome engineering.
ContributorsStandage-Beier, Kylie S (Author) / Wang, Xiao (Thesis advisor) / Brafman, David A (Committee member) / Tian, Xiao-jun (Committee member) / Arizona State University (Publisher)
Created2018
Description
Synthetic biology is an emerging field which melds genetics, molecular biology, network theory, and mathematical systems to understand, build, and predict gene network behavior. As an engineering discipline, developing a mathematical understanding of the genetic circuits being studied is of fundamental importance. In this dissertation, mathematical concepts for understanding, predicting, and controlling gene transcriptional networks are presented and applied to two synthetic gene network contexts. First, this engineering approach is used to improve the function of the guide ribonucleic acid (gRNA)-targeted, dCas9-regulated transcriptional cascades through analysis and targeted modification of the RNA transcript. In so doing, a fluorescent guide RNA (fgRNA) is developed to more clearly observe gRNA dynamics and aid design. It is shown that through careful optimization, RNA Polymerase II (Pol II) driven gRNA transcripts can be strong enough to exhibit measurable cascading behavior, previously only shown in RNA Polymerase III (Pol III) circuits. Second, inherent gene expression noise is used to achieve precise fractional differentiation of a population. Mathematical methods are employed to predict and understand the observed behavior, and metrics for analyzing and quantifying similar differentiation kinetics are presented. Through careful mathematical analysis and simulation, coupled with experimental data, two methods for achieving ratio control are presented, with the optimal schema for any application being dependent on the noisiness of the system under study. Together, these studies push the boundaries of gene network control, with potential applications in stem cell differentiation, therapeutics, and bio-production.
ContributorsMenn, David J (Author) / Wang, Xiao (Thesis advisor) / Kiani, Samira (Committee member) / Haynes, Karmella (Committee member) / Nielsen, David (Committee member) / Marshall, Pamela (Committee member) / Arizona State University (Publisher)
Created2018
Description
Synthetic manipulation of chromatin dynamics has applications for medicine, agriculture, and biotechnology. However, progress in this area requires the identification of design rules for engineering chromatin systems. In this thesis, I discuss research that has elucidated the intrinsic properties of histone binding proteins (HBP), and apply this knowledge to engineer novel chromatin binding effectors. Results from the experiments described herein demonstrate that the histone binding domain from chromobox protein homolog 8 (CBX8) is portable and can be customized to alter its endogenous function. First, I developed an assay to identify engineered fusion proteins that bind histone post translational modifications (PTMs) in vitro and regulate genes near the same histone PTMs in living cells. This assay will be useful for assaying the function of synthetic histone PTM-binding actuators and probes. Next, I investigated the activity of a novel, dual histone PTM binding domain regulator called Pc2TF. I characterized Pc2TF in vitro and in cells and show it has enhanced binding and transcriptional activation compared to a single binding domain fusion called Polycomb Transcription Factor (PcTF). These results indicate that valency can be used to tune the activity of synthetic histone-binding transcriptional regulators. Then, I report the delivery of PcTF fused to a cell penetrating peptide (CPP) TAT, called CP-PcTF. I treated 2D U-2 OS bone cancer cells with CP-PcTF, followed by RNA sequencing to identify genes regulated by CP-PcTF. I also showed that 3D spheroids treated with CP-PcTF show delayed growth. This preliminary work demonstrated that an epigenetic effector fused to a CPP can enable entry and regulation of genes in U-2 OS cells through DNA independent interactions. Finally, I described and validated a new screening method that combines the versatility of in vitro transcription and translation (IVTT) expressed protein coupled with the histone tail microarrays. Using Pc2TF as an example, I demonstrated that this assay is capable of determining binding and specificity of a synthetic HBP. I conclude by outlining future work toward engineering HBPs using techniques such as directed evolution and rational design. In conclusion, this work outlines a foundation to engineer and deliver synthetic chromatin effectors.
ContributorsTekel, Stefan (Author) / Haynes, Karmella (Thesis advisor) / Mills, Jeremy (Committee member) / Caplan, Michael (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2019
Description
How do you convey what’s interesting and important to you as an artist in a digital world of constantly shifting attentions? For many young creatives, the answer is original characters, or OCs. An OC is a character that an artist creates for personal enjoyment, whether based on an already existing story or world, or completely from their own imagination.
As creations made for purely personal interests, OCs are an excellent elevator pitch to talk one creative to another, opening up opportunities for connection in a world where communication is at our fingertips but personal connection is increasingly harder to make. OCs encourage meaningful interaction by offering themselves as muses, avatars, and story pieces, and so much more, where artists can have their characters interact with other creatives through many different avenues such as art-making, table top games, or word of mouth.
In this thesis, I explore the worlds and aesthetics of many creators and their original characters through qualitative research and collaborative art-making. I begin with a short survey of my creative peers, asking general questions about their characters and thoughts on OCs, then move to sketching characters from various creators. I focus my research to a group of seven core creators and their characters, whom I interview and work closely with in order to create a series of seven final paintings of their original characters.
As creations made for purely personal interests, OCs are an excellent elevator pitch to talk one creative to another, opening up opportunities for connection in a world where communication is at our fingertips but personal connection is increasingly harder to make. OCs encourage meaningful interaction by offering themselves as muses, avatars, and story pieces, and so much more, where artists can have their characters interact with other creatives through many different avenues such as art-making, table top games, or word of mouth.
In this thesis, I explore the worlds and aesthetics of many creators and their original characters through qualitative research and collaborative art-making. I begin with a short survey of my creative peers, asking general questions about their characters and thoughts on OCs, then move to sketching characters from various creators. I focus my research to a group of seven core creators and their characters, whom I interview and work closely with in order to create a series of seven final paintings of their original characters.
ContributorsCote, Jacqueline (Author) / Button, Melissa M (Thesis director) / Dove-Viebahn, Aviva (Committee member) / School of Art (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
In the past ten years, the United States’ sound recording industries have experienced significant decreases in employment opportunities for aspiring audio engineers from economic imbalances in the music industry’s digital streaming era and reductions in government funding for career and technical education (CTE). The Recording Industry Association of America reports promises of music industry sustainability based on increasing annual revenues in paid streaming services and artists’ high creative demand. The rate of new audio engineer entries in the sound recording subsection of the music industry is not viable to support streaming artists’ high demand to engineer new music recordings. Offering CTE programs in secondary education is rare for aspiring engineers with insufficient accessibility to pursue a post-secondary or vocational education because of financial and academic limitations. These aspiring engineers seek alternatives for receiving an informal education in audio engineering on the Internet using video sharing services like YouTube to search for tutorials and improve their engineering skills. The shortage of accessible educational materials on the Internet restricts engineers from advancing their own audio engineering education, reducing opportunities to enter a desperate job market in need of independent, home studio-based engineers. Content creators on YouTube take advantage of this situation and commercialize their own video tutorial series for free and selling paid subscriptions to exclusive content. This is misleading for newer engineers because these tutorials omit important understandings of fundamental engineering concepts. Instead, content creators teach inflexible engineering methodologies that are mostly beneficial to their own way of thinking. Content creators do not often assess the incompatibility of teaching their own methodologies to potential entrants in a profession that demands critical thinking skills requiring applied fundamental audio engineering concepts and techniques. This project analyzes potential solutions to resolve the deficiencies in online audio engineering education and experiments with structuring simple, deliverable, accessible educational content and materials to new entries in audio engineering. Designing clear, easy to follow material to these new entries in audio engineering is essential for developing a strong understanding for the application of fundamental concepts in future engineers’ careers. Approaches to creating and designing educational content requires translating complex engineering concepts through simplified mediums that reduce limitations in learning for future audio engineers.
ContributorsBurns, Triston Connor (Author) / Tobias, Evan (Thesis director) / Libman, Jeff (Committee member) / Department of Information Systems (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05