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- Creators: Arizona State University
- Creators: Brafman, David
- Creators: Harrington Bioengineering Program
- Status: Published
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
Most people are experts in some area of information; however, they may not be knowledgeable about other closely related areas. How knowledge is generalized to hierarchically related categories was explored. Past work has found little to no generalization to categories closely related to learned categories. These results do not fit well with other work focusing on attention during and after category learning. The current work attempted to merge these two areas of by creating a category structure with the best chance to detect generalization. Participants learned order level bird categories and family level wading bird categories. Then participants completed multiple measures to test generalization to old wading bird categories, new wading bird categories, owl and raptor categories, and lizard categories. As expected, the generalization measures converged on a single overall pattern of generalization. No generalization was found, except for already learned categories. This pattern fits well with past work on generalization within a hierarchy, but do not fit well with theories of dimensional attention. Reasons why these findings do not match are discussed, as well as directions for future research.
ContributorsLancaster, Matthew E (Author) / Homa, Donald (Thesis advisor) / Glenberg, Arthur (Committee member) / Chi, Michelene (Committee member) / Brewer, Gene (Committee member) / Arizona State University (Publisher)
Created2013
Description
A new loop configuration capable of reducing power radiation magnitudes lower than conventional loops has been developed. This configuration is demonstrated for the case of two coaxial loops of 0.1 meter radius coupled via the magnetic reactive field. Utilizing electromagnetism theory, techniques from antenna design and a new near field design initiative, the ability to design a magnetic field has been investigated by using a full wave simulation tool. The method for realization is initiated from first order physics model, ADS and onto a full wave situation tool for the case of a non-radiating helical loop. The exploration into the design of a magnetic near field while mitigating radiation power is demonstrated using an real number of twists to form a helical wire loop while biasing the integer twisted loop in a non-conventional moebius termination. The helix loop setup as a moebius loop convention can also be expressed as a shorted antenna scheme. The 0.1 meter radius helix antenna is biased with a 1MHz frequency that categorized the antenna loop as electrically small. It is then demonstrated that helical configuration reduces the electric field and mitigates power radiation into the far field. In order to compare the radiated power reduction performance of the helical loop a shielded loop is used as a baseline for comparison. The shielded loop system of the same geometric size and frequency is shown to have power radiation expressed as -46.1 dBm. The power radiated mitigation method of the helix loop reduces the power radiated from the two loop system down to -98.72 dBm.
ContributorsMoreno, Fernando (Author) / Diaz, Rodolfo (Thesis advisor) / Aberle, James T., 1961- (Committee member) / Kozicki, Michael (Committee member) / Arizona State University (Publisher)
Created2015
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
The work described in the thesis involves the synthesis of a molecular triad which is designed to undergo proton coupled electron transfer (PCET) upon irradiation with light. Photoinduced PCET is an important process that many organisms use and the elucidation of its mechanism will allow further understanding of this process and its potential applications. The target compound designed for PCET studies consists of a porphyrin chromophore (also a primary electron donor), covalently linked to a phenol-imidazole (secondary electron donor), and a C60 (primary electron acceptor). The phenol-imidazole moiety of this system is modeled after the TyrZ His-190 residues in the reaction center of Photosystem II (PS II). These residues participate in an intermolecular H-bond between the phenol side chain of TyrZ and the imidazole side chain of His-190. The phenol side chain of TyrZ is the electron transfer mediator between the oxygen evolving complex (OEC) and P680 (primary electron donor) in PSII. During electron transfer from TyrZ to P680*+, the phenolic proton of TyrZ becomes highly acidic (pKa~-2) and the hydrogen is preferentially transferred to the relatively basic imidazole of His-190 through a pre-existing hydrogen bond. This PCET process avoids a charged intermediate, on TyrZ, and results in a neutral phenolic radical (TyrZ*). The current research consists of building a molecular triad, which can mimic the photoinduced PCET process of PSII. The following, documents the synthetic progress in the synthesis of a molecular triad designed to investigate the mechanism of PCET as well as gain further insight on how this process can be applied in artificial photosynthetic devices.
ContributorsPatterson, Dustin (Author) / Moore, Ana L (Thesis advisor) / Gust, Devens (Committee member) / Skibo, Edward B (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
The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial proteins expressed in human cell lines, yet they exhibit an organizing principle: that genes and proteins may be treated as modular units that can be moved from their native organism to a novel one. However, protein behavior is always unpredictable; drop-in functionality is not guaranteed.
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
ContributorsDaer, René (Author) / Haynes, Karmella (Thesis advisor) / Brafman, David (Committee member) / Nielsen, David (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2017