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- Creators: Arizona State University
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
This dissertation offers three essays that investigate consumers’ health-related food choices and behaviors from three different, yet complementary, angles. The first essay uses an eye-tracking experiment to examine consumers’ visual attention to the Nutrition Facts Panels for healthy and unhealthy products. In this essay, I focus on how involvement and familiarity affect consumers’ attention toward the Nutrition Facts panel and how these two psychological factors interact with new label format changes in attracting consumers’ attention. In the second essay, I demonstrate using individual-level scanner data that nutritional attributes interact with marketing mix elements to affect consumers’ nutrition intake profiles and their intra-category substitution patterns. My findings suggest that marketing-mix sensitivities are correlated with consumers’ preferences for nutrient attributes in ways that depend on the “healthiness” of the nutrient. For instance, featuring promotes is positively correlated with “healthy” nutritional characteristics such as high-protein, low-fat, or low-carbohydrates, whereas promotion and display are positively correlated with preferences for “unhealthy” characteristics such as high-fat, or high-carbohydrates. I use model simulations to show that some marketing-mix elements are able to induce consumers to purchase items with higher maximum-content levels than others. The fourth chapter shows that dieters are not all the same. I develop and validate a new scale that measures lay theories about abstinence vs. moderation. My findings from a series of experiments indicate that dieters’ recovery from recalled vs. actual indulgences depend on whether they favor abstinence or moderation. However, compensatory coping strategies provide paths for people with both lay theories to recover after an indulgence, in their own ways. The three essays provide insights into individual differences that determine approaches of purchase behaviors, and consumption patterns, and life style that people choose, and these insights have potential policy implications to aid in designing the food-related interventions and policies to improve the healthiness of consumers’ consumption profiles and more general food well-being.
ContributorsXie, Yi (Author) / Richards, Timothy (Thesis advisor) / Mandel, Naomi (Committee member) / Grebitus, Carola (Committee member) / Arizona State University (Publisher)
Created2018
Description
This article can be divided into six parts.
The first chapter analyzes the background, theatrical and particle reasons of this research. The author argues that the management of law firm needs a set of good system. The first one is operating the law firm in scale, and the other on is corporate management model, which shall be constructed in detail in the paper and will be put into practice by the law firm in which the author is worked.
The second chapter will introduce modern management theory, combining the situation of management in our law firm to analyze, raising some reasonable suggestions and instructions to promote our law firm to achieve the corporate management.
In the third chapter, the first chapter, starting with the review of the development process of foreign and our law firms, listing the organizational forms and the characteristics of our law firm, analyzing the situation and the drawbacks of the law firm management.
The fourth and fifth chapter introduce he background, the connotation of the corporate management model, listing the development and successful experience of some typical cases in respect of corporate management.
In the last chapter, the construction of corporate management model will be introduced in terms of organization form, human resource management and informationizing development.
The corporate management model is not mature in china. Though it is not easy to reform the existing model, but it should be believed that the development benefiting the legal industry will be achieved.
The first chapter analyzes the background, theatrical and particle reasons of this research. The author argues that the management of law firm needs a set of good system. The first one is operating the law firm in scale, and the other on is corporate management model, which shall be constructed in detail in the paper and will be put into practice by the law firm in which the author is worked.
The second chapter will introduce modern management theory, combining the situation of management in our law firm to analyze, raising some reasonable suggestions and instructions to promote our law firm to achieve the corporate management.
In the third chapter, the first chapter, starting with the review of the development process of foreign and our law firms, listing the organizational forms and the characteristics of our law firm, analyzing the situation and the drawbacks of the law firm management.
The fourth and fifth chapter introduce he background, the connotation of the corporate management model, listing the development and successful experience of some typical cases in respect of corporate management.
In the last chapter, the construction of corporate management model will be introduced in terms of organization form, human resource management and informationizing development.
The corporate management model is not mature in china. Though it is not easy to reform the existing model, but it should be believed that the development benefiting the legal industry will be achieved.
ContributorsZhu, Ping (Author) / Gu, Bin (Thesis advisor) / Chang, Chun (Thesis advisor) / Zhu, Ning (Committee member) / Arizona State University (Publisher)
Created2017
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
Due to the booming young mothers and fathers in the new era as well as the changes in the concept of parenting and the favorable liberalization of China's second child policy, the maternal-baby nursing market continues to grow, and it has become a must for businesses nowadays. In 2020, the size of the maternal-baby nursing market will reach 3.6 trillion. (Data: Yibang Power China's Maternal and Child Industry White Paper 2017).
The rapid development of mobile Internet, highly transparent information, consumers grasp the sovereignty, along with the rise of the middle class, consumption increase encouraged personalized, customized needs. The boundaries between online and offline are becoming increasingly blurred. Consumers are more inclined to choose multi-category with service channel providers. If the retailers still rely on good market resources, and the difference between the sales and purchase of commodities, they will face a huge challenge of the decrease in passenger flow and a decline in performance.
The paper takes the relationship between maternal-baby nursing retailers and targets customers as the study object, based on customer service of maternal-baby nursing retailer data, empirical studies, we found that this particular group, mothers and babies, especially value safety, quality, public praise and community review. If the retail enterprise attaches importance to establishing relationships with customers and enhances the relational viscosity through mutual trust, emotional formation and spread of public praise, it will help to increase the traffic volume and increase the output value of single customers.
The maternal-baby nursing retailers form a strong relationship between enterprises and customers by establishing a strong relationship between products and customers, employees and customers, and customers to customers. Maternal-baby nursing retailers operate single-customer value deeply, build a heavy membership system and manage customer assets, thereby enhancing their brand and performance.
The research on the method of establishing the strong tie can be considered as an analysis of feasibility. The research results of this paper will help to improve the overall customer service experience and satisfaction of the mother and infant retail industry, enhance the development of the whole industry and draw significance lessons from other service industries.
The rapid development of mobile Internet, highly transparent information, consumers grasp the sovereignty, along with the rise of the middle class, consumption increase encouraged personalized, customized needs. The boundaries between online and offline are becoming increasingly blurred. Consumers are more inclined to choose multi-category with service channel providers. If the retailers still rely on good market resources, and the difference between the sales and purchase of commodities, they will face a huge challenge of the decrease in passenger flow and a decline in performance.
The paper takes the relationship between maternal-baby nursing retailers and targets customers as the study object, based on customer service of maternal-baby nursing retailer data, empirical studies, we found that this particular group, mothers and babies, especially value safety, quality, public praise and community review. If the retail enterprise attaches importance to establishing relationships with customers and enhances the relational viscosity through mutual trust, emotional formation and spread of public praise, it will help to increase the traffic volume and increase the output value of single customers.
The maternal-baby nursing retailers form a strong relationship between enterprises and customers by establishing a strong relationship between products and customers, employees and customers, and customers to customers. Maternal-baby nursing retailers operate single-customer value deeply, build a heavy membership system and manage customer assets, thereby enhancing their brand and performance.
The research on the method of establishing the strong tie can be considered as an analysis of feasibility. The research results of this paper will help to improve the overall customer service experience and satisfaction of the mother and infant retail industry, enhance the development of the whole industry and draw significance lessons from other service industries.
ContributorsWang, Jianguo (Author) / Gu, Bin (Thesis advisor) / Chen, Hong (Thesis advisor) / Cui, Haitao (Committee member) / Arizona State University (Publisher)
Created2018
Description
This study examines the 3 key questions of media budget allocation, to find our a better invest model. Including spending share of traditional media and digital media, program selection strategy and duration mix optimization to analyse the trend of sample A (a global cosmetic brand) . Based on every test media campaign, we do research of media performance and sales volumn, add youth consumer behavior result, to develop a media investment ROI model for this brand. Create the evaluation system according to past big data and find the learnings of different length TVC usage. Of course all relavant findings and implications will be summarized after every section.
ContributorsXu, Jin (Author) / Gu, Bin (Thesis advisor) / Chen, Xinlei (Thesis advisor) / Shao, Benjamin (Committee member) / Arizona State University (Publisher)
Created2018