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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
We created an Android application, Impromp2, which allows users to search for and save events of interest to them in the Phoenix area. The backend, built on the Parse platform, gathers events daily using Web services and stores them in a database. Impromp2 was designed to improve upon similarly-purposed apps available for Android devices in several key ways, especially in user interface design and data interaction capability. This is a full-stack software project that explores databases and their performance considerations, Web services, user interface design, and the challenges of app development for a mobile platform.
ContributorsNorth, Joseph Robert (Author) / Balasooriya, Janaka (Thesis director) / Nakamura, Mutsumi (Committee member) / Faucon, Philippe (Committee member) / Barrett, The Honors College (Contributor) / Computer Science and Engineering Program (Contributor) / School of Mathematical and Statistical Sciences (Contributor)
Created2015-05
Description
"The Legal Adventures of Frankie and Rosie" is a creative project that explores the nontraditional format of comics to express creative nonfiction. The project is a set of 30 independent comics that focuses on two primary college-going students who are based off of the authors. The characters, Frankie and Rosie narrate their stories through dialogue. The authors use this narrative model to archive their college experience at ASU. Representing creative nonfiction through comics yields an amalgamated format that can be challenging for both the writers to produce as well as for the readers to consume. Ultimately, the project serves as an attempt to test whether or not the comic medium can stand by itself as an appropriate format to express creative nonfictional narratives without becoming a diluted combination of its purer predecessors.
ContributorsBennewitz, Dana (Co-author) / Ray, Payel (Co-author) / Martin, Thomas (Thesis director) / Scott Lynch, Jacquelyn (Committee member) / Civil, Environmental and Sustainable Engineering Programs (Contributor) / Barrett, The Honors College (Contributor) / School of Geographical Sciences and Urban Planning (Contributor) / Electrical Engineering Program (Contributor) / School of Social Transformation (Contributor) / School of Mathematical and Statistical Sciences (Contributor)
Created2015-05
Description
Rainbow Connection is an integrated choir with members on and off the autism spectrum. It was founded in the spring of 2012 by Barrett students Ali Friedman, Megan Howell, and Victoria Gilman as part of an honors thesis creative project. Rainbow Connection uses the rehearsal process and other creative endeavors to foster natural relationship building across social gaps. A process-oriented choir, Rainbow Connection's main goals concern the connections made throughout the experience rather than the final musical product. The authors believe that individual, non-hierarchical relationships are the keys to breaking down systemized gaps between identity groups and that music is an ideal facilitator for fostering such relationships. Rainbow Connection operates under the premise that, like colors in a rainbow, choir members create something beautiful not by melding into one homogenous group, but by collaboratively showcasing their individual gifts. This paper will highlight the basic premise and structure of Rainbow Connection, outline the process of enacting the choir, and describe the authors' personal reactions and takeaways from the project.
ContributorsFriedman, Alexandra (Co-author) / Gilman, Victoria (Co-author) / Howell, Megan (Co-author) / Rio, Robin (Thesis director) / Schildkret, David (Committee member) / Barrett, The Honors College (Contributor) / School of Music (Contributor) / School of Mathematical and Statistical Sciences (Contributor)
Created2014-12
Description
The title means nothing because the stories have little in common, aside from the fact that I wrote them. The common theme of anxiety was unintentional, though it is prevalent in the stories, poetry and my life. Each story is written from a different style, with a different interest in mind. The poetry that breaks up the stories is mine, and also free of common bonds. People whom I love inspired some of them; others stem from people with whom I was (or still am) angry. Some of them are just me trying to write poetry like other successful poets, who seem to know something I don't. I wrote this set of stories and poems because I wanted to see if I could do it. I wanted to challenge myself in a new medium (two new mediums really, if you separate literature and poetry). I wanted to prove to myself that I could do it, if I really set my mind to it. I wanted to have some wealth of words, which I could record myself reading. Overall, I hope that you enjoy these stories and words. I wrote them to entertain myself, and they seem to do that pretty well. If you don't like them, stop reading. If you do like them, keep reading and tell everyone you know about this collection. I'm proud of my work here, so anything beyond that is icing on my cake.
ContributorsRagatz, Zachariah Edward (Author) / Scott, Jason Davids (Thesis director) / Espinosa, Micha (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / School of Film, Dance and Theatre (Contributor)
Created2015-05
Description
Transgene expression in mammalian cells has been shown to meet resistance in the form of silencing due to chromatin buildup within the cell. Interactions of proteins with chromatin modulate gene expression profiles. Synthetic Polycomb transcription factor (PcTF) variants have the potential to reactivate these silence transgenes as shown in Haynes & Silver 2011. PcTF variants have been constructed via TypeIIS assembly to further investigate this ability to reactive transgenes. Expression in mammalian cells was confirmed via fluorescence microscopy and red fluorescent protein (RFP) expression in cell lysate. Examination of any variation in conferment of binding strength of homologous Polycomb chromodomains (PCDs) to its trimethylated lysine residue target on histone three (H3K27me3) was investigated using a thermal shift assay. Results indicate that PcTF may not be a suitable protein for surveying with SYPRO Orange, a dye that produces a detectable signal when exposed to the hydrophobic domains of the melting protein. A cell line with inducible silencing of a chemiluminescent protein was used to determine the effects PcTF variants had on gene reactivation. Results show down-regulation of the target reporter gene. We propose this may be due to PcTF not binding to its target; this would cause PcTF to deplete transcriptional machinery in the nucleus. Alternatively, the CMV promoter could be sequestering transcriptional machinery in its hyperactive transcription of PcTF leading to widespread down-regulation. Finally, the activation domain used may not be appropriate for this cell type. Future PcTF variants will address these hypotheses by including multiple Polycomb chromodomains (PCDs) to alter the binding dynamics of PcTF to its target, and by incorporating alternative promoters and activation domains.
ContributorsGardner, Cameron Lee (Author) / Haynes, Karmella (Thesis director) / Stabenfeldt, Sarah (Committee member) / Barrett, The Honors College (Contributor) / Department of Finance (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
Description
Since the acceptance of Einstein's special theory of relativity by the scientific community, authors of science fiction have used the concept of time dilation to permit seemingly impossible feats. Simple spacecraft acceleration schemes involving time dilation have been considered by scientists and fiction writers alike. Using an original Java program based upon the differential equations for special relativistic kinematics, several scenarios for round trip excursions at relativistic speeds are calculated and compared, with particular attention to energy budget and relativistic time passage in all relevant frames.
ContributorsAlfson, Jonathan William (Author) / Jacob, Richard (Thesis director) / Covatto, Carl (Committee member) / Foy, Joseph (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Physics (Contributor)
Created2015-05
Description
Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules that modify gene expression of a designated receiver cell. While useful, these three quorum sensing pathways exhibit a nontrivial level of crosstalk, hindering robust engineering and leading to unexpected effects in a given design. To address the lack of orthogonality among these three quorum sensing pathways, previous scientists have attempted to perform directed evolution on components of the quorum sensing pathway. While a powerful tool, directed evolution is limited by the subspace that is defined by the protein. For this reason, we take an evolutionary biology approach to identify new orthogonal quorum sensing networks and test these networks for cross-talk with currently-used networks. By charting characteristics of acyl homoserine lactone (AHL) molecules used across quorum sensing pathways in nature, we have identified favorable candidate pathways likely to display orthogonality. These include Aub, Bja, Bra, Cer, Esa, Las, Lux, Rhl, Rpa, and Sin, which we have begun constructing and testing. Our synthetic circuits express GFP in response to a quorum sensing molecule, allowing quantitative measurement of orthogonality between pairs. By determining orthogonal quorum sensing pairs, we hope to identify and adapt novel quorum sensing pathways for robust use in higher-order genetic circuits.
ContributorsMuller, Ryan (Author) / Haynes, Karmella (Thesis director) / Wang, Xiao (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
This creative project centers on creating evaluative writing about film, in the form of a film review blog. Preliminary writing was done, in which the distinction was made between critical film writing and movie reviewing, as well as an analysis of how film critics have honed in their criticism and what makes their content effective for their audience. The rest of the writing for this project consists of a total of 15 reviews for 15 different movies released in 2017 and 2018. In these reviews, there is a brief introduction of the plot and context in which the film is made, followed by an evaluative analysis of what made the film effective or ineffective in achieving its artistic goals. The reviews involve an amalgamation of the content and topics taught in the Film and Media Studies program at Arizona State University, from screenwriting to cinematography. This process of writing reviews and being edited by the Director and Second Reader allows for the opportunity to find a unique writing voice and create content that is accessible for the wide audience that would be reading the work. All of the writing completed for this project (except for the "My Favorite Film Critics" piece) is compiled together in a WordPress blog, in an easily readable and accessible format. The blog itself serves as a way to reach the desired audience, as well as entice them to engage with the writing and the films being written about. This includes providing images and trailers for each respective film, to add a visual component to the writing. The final product is a unique way to engage with the content taught in the Film and Media Studies program, while simultaneously building a portfolio of writing that will be expanded upon and continued in the future.
ContributorsPolich, Brennan Taylor (Author) / Green, Michael (Thesis director) / Bernstein, Gregory (Committee member) / Department of English (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05