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Description
Metabolic engineering is an extremely useful tool enabling the biosynthetic production of commodity chemicals (typically derived from petroleum) from renewable resources. In this work, a pathway for the biosynthesis of styrene (a plastics monomer) has been engineered in Escherichia coli from glucose by utilizing the pathway for the naturally occurring amino acid phenylalanine, the precursor to styrene. Styrene production was accomplished using an E. coli phenylalanine overproducer, E. coli NST74, and over-expression of PAL2 from Arabidopsis thaliana and FDC1 from Saccharomyces cerevisiae. The styrene pathway was then extended by just one enzyme to either (S)-styrene oxide (StyAB from Pseudomonas putida S12) or (R)-1,2-phenylethanediol (NahAaAbAcAd from Pseudomonas sp. NCIB 9816-4) which are both used in pharmaceutical production. Overall, these pathways suffered from limitations due to product toxicity as well as limited precursor availability. In an effort to overcome the toxicity threshold, the styrene pathway was transferred to a yeast host with a higher toxicity limit. First, Saccharomyces cerevisiae BY4741 was engineered to overproduce phenylalanine. Next, PAL2 (the only enzyme needed to complete the styrene pathway) was then expressed in the BY4741 phenylalanine overproducer. Further strain improvements included the deletion of the phenylpyruvate decarboxylase (ARO10) and expression of a feedback-resistant choristmate mutase (ARO4K229L). These works have successfully demonstrated the possibility of utilizing microorganisms as cellular factories for the production styrene, (S)-styrene oxide, and (R)-1,2-phenylethanediol.
ContributorsMcKenna, Rebekah (Author) / Nielsen, David R (Thesis advisor) / Torres, Cesar (Committee member) / Caplan, Michael (Committee member) / Jarboe, Laura (Committee member) / Haynes, Karmella (Committee member) / Arizona State University (Publisher)
Created2014
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
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
The purpose of this paper is to understand how companies are finding high potential employees and if they are leaving top talent behind in their approach. Eugene Burke stated in 2014 that 55% of employees that are labeled as a High Potential Employee will turn over and move companies. Burke (2014) also states that the average high potential employee tenure is five years. The Corporate Leadership Council says that on average, 27% of a company's development budget is spent on its high potential program (CEB 2017). For a midsize company, the high potential development budget is almost a million dollars for only a handful of employees, only to see half of the investment walking out the door to another company . Furthermore, the Corporate Leadership Council said that a study done in 2005 revealed that 50% of high potential employees had significant problems within their job (Kotlyar and Karkowsky 2014). Are time and resources are being given to the wrong employees and the right employees are being overlooked? This paper exams how companies traditionally select high potential employees and where companies are potentially omitting employees who would be better suited for the program. This paper proposes that how a company discovers their top talent will correlate to the number of turnovers or struggles that a high potential employee has on their job. Future research direction and practical considerations are also presented in this paper.
ContributorsHarrison, Carrie (Author) / Mizzi, Philip (Thesis director) / Ruediger, Stefan (Committee member) / Department of Management and Entrepreneurship (Contributor) / School of Sustainability (Contributor) / Department of Supply Chain Management (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
In which industry that has ever been profit generating, does a firm profit from their failure? The United States has a mass incarceration problem. With 25% of the world prison population residing in the US, spending on detention costs the US government $80 billion annually. Over 50% of the individuals incarcerated in America are of black or Latino descent. This massive growth in the incarcerated population of America began in the 1970s and with the passive support of American citizens has created an industry whose players profit from the detention of people. Currently, the privately run detention facilities in the United States hold 7% of state prisoners, 18% of federal prisoners, and nearly 75% of ICE's undocumented detainee population. The detention of people for profit is an idea rooted in the same profit motive that allowed the institution of slavery to flourish. However even after the 13th Amendment abolished slavery in the U.S., the oppressive forces behind slave-era economics have been perpetuated through legislation and policies that continued the stratification of society and reinforcement of the social order. With the help of corporate lobbyists, political action committees, and organizations such as the American Legislative Exchange Council, the corporate shareholders of private prisons, such as CoreCivic and The GEO Group, are able to directly align their profit-driven interests with those of federal and state legislators. By the incorporation of legislation and policy into state and federal law, the shareholders of private prisons are able to directly affect legislation as well as their own potential for profit. The justification for the usage of private prisons is thought to be seen in the price savings and flexibility that it provides for federal and state governments. However, due to the law enforcement contractor's exemption from public record laws, there is no clear evidence of where the cost savings occur, or even if there are cost savings at all. Is it ethical for a for-profit-prison corporation to be responsible for the care, security, and rehabilitation of an individual, when if they fail to rehabilitate the individual, it will add to the number of inmates under their control? The measure of a prison's failure to rehabilitate an inmate is considered the recidivism rate, and is affected when an inmate leaves a detention facility, commits another crime, then is arrested. This profit motive is causing our society to incarcerate increasing numbers of people in private prisons. For-profit prisons financially benefit from long-term incarceration and recidivism. The passive investments from public and private employees and institutions through investment corporations are the legs that allow the private prison industry to stand. Twenty-nine investment firms, such as The Vanguard Group and Fidelity Investments, own nearly two-thirds of the two largest players in the private prison industry. This includes the passive investments by public institutions such as the Arizona State University Foundation's $600 million endowment fund as well as the $500 million directly invested into CoreCivic and GEO Group from the University of Texas/ Texas A&M Investment Management Company. The goal of abolishing private prisons will require years of litigation against the giants of the industry as well as the governmental entities supporting them. However, we can start today by demanding divestiture by our school and similar institutions as well continuing to share the knowledge of the oppressive forces associated with the detention of individuals for profit.
ContributorsBayham, Michael (Author) / Gomez, Alan (Thesis director) / Dacey, John (Committee member) / W.P. Carey School of Business (Contributor) / Department of Supply Chain Management (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The purpose of this thesis is to explore how Blockchain technology can help solve problems large corporations commonly face. For example, it is a common problem for large businesses and organizations to manage sales contracts with thousands of items on them. Likewise, it can be difficult to accurately monitor complex payment histories with thousands of items on them. Another issue is the difficulty that is introduced when making periodic reconciliations based on separate recording systems. At a broader level, some organizations may hesitate to do business with new strange companies or oversea companies for the first time because they do not trust that the other organization can deliver what they promise. Such problems cost organizations a lot of money, effort, and time to solve. However, Blockchain technology, first developed in 2009, could revolutionize how the business community deals with these common problems. The shared and immutable ledger on Blockchain can help organizations to keep track on transactions, manage the contracts in a smarter way, ensure correct purchase history records, eliminate the periodically reconciliation processes, and provide visibility for real-time transactions.
ContributorsHuynh, Phu Thanh (Author) / Popova, Laura (Thesis director) / Pankaj, Sneha (Committee member) / Department of Supply Chain Management (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
As climate change and air pollution continue to plague the world today, committed citizens are doing their part to minimize their environmental impact. However, financial limitations have hindered a majority of individuals from adopting clean, renewable energy such as rooftop photovoltaic solar systems. England Sustainability Consulting plans to reverse this limitation and increase affordability for residents across Northern California to install solar panel systems for their energy needs. The purpose of this proposal is to showcase a new approach to procuring solar panel system components while offering the same products needed by each customer. We will examine market data to further prove the feasibility of this business approach while remaining profitable and spread our company's vision across all of Northern California.
ContributorsEngland, Kaysey (Author) / Dooley, Kevin (Thesis director) / Keahey, Jennifer (Committee member) / Department of Supply Chain Management (Contributor) / School of Social and Behavioral Sciences (Contributor) / W.P. Carey School of Business (Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The National Basketball Association (NBA) is one of the Big Four Sporting Leagues of US Professional Sports. In recent years, the NBA has enjoyed milestone seasons in both attendance and television ratings, resulting in steady increases to both, over the previous decade. (Morgan, 2017) This surge can be attributed in part to the integration of "cultural recognition" initiatives and the overall message of inclusivity on the part of NBA franchises, with their respective promotions and advertisements such as television, social media, radio, etc. Heritage Nights, such as "Noche Latina," among other variants in the NBA, typically feature culturally influenced changes to team logos, giveaways, and other consumer offerings. In markets where Hispanics make up a significant percentage of the fan-base, such as Phoenix, NBA franchises such as the Phoenix Suns must ascertain the financial or perceptual impacts, associated with risks of stereotyping, offending or otherwise unintentionally alienating different categories of fans. To this end, data was collected from the local NBA franchises' fanbase, specifically Phoenix Suns season-ticket holders, and was statistically checked for significant relationships between both categories of fans and several different variables. This analysis found that only $192K in revenue is being missed through the investment of Heritage Nights, and that fan perceptions of stereotypical or offensive giveaways and practices have no significant effect on game or event attendance, despite the stereotypes toward giveaways and practices still being present. Implications of this study provide possible next steps for the Suns and continue to widen the scope of demographical sports marketing both in professional basketball and beyond.
ContributorsGibbens, Patrick Alexander (Author) / Eaton, John (Thesis director) / McIntosh, Daniel (Committee member) / Department of Supply Chain Management (Contributor) / School of Music (Contributor) / Department of Marketing (Contributor) / W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Sagebrush Coffee is a small business in Chandler, Arizona that purchases green beans, roasts them in small batches for quality, and ships fresh, gourmet roasted coffee beans across the nation. Deciding which coffee beans to buy and roast is one of the most crucial business decisions Sagebrush and other gourmet coffee roasters face. Further complicating this decision is the fact that coffee is a crop, and like all crops, has a specific growing season and the exact same product cannot usually be ordered from year to year, even if it proves to be successful. The goal of this research is to use data analytics and visualization to help Sagebrush make better purchasing decisions by identifying consumer purchasing trends and providing a recommendation for their portfolio mix. In the end, I found that Latin American coffees are popular with both returning and first-time customers, but a specific country of origin does not appear to be associated with the top coffee producing countries. Additionally, December is a critical month for Sagebrush and Sagebrush should make sure to target the states with the most sales: California, Pennsylvania, and New York. Arizona has growth potential as it is not one of the top three locations, despite the presence of a physical store. Also included in the following report is a portfolio recommendation suggesting how many of each product based on region, processing type, and roast level to carry in inventory.
ContributorsBlue, Jessica Morgan (Author) / Kellso, James (Thesis director) / Davila, Eddie (Committee member) / Department of Information Systems (Contributor) / Economics Program in CLAS (Contributor) / Department of Supply Chain Management (Contributor) / Morrison School of Agribusiness (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05