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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
"Many Faces" is the result of a year-long exploration of online harassment. It includes multiple graphic design projects which reference the phenomenon of online harassment and attempt to solve it (or at least contribute to a solution), all in different ways.
According to a survey performed by Pew Research in 2014, 40% of Internet users have experienced online harassment. 18% had experienced severe harassment – stalking, sexual harassment, physical threats – while 22% had only experienced less severe harassment, such as name-calling. Women ages 18–24 receive a disproportionately large percentage of all severe online harassment. The emotional trauma suffered from severe or long-term harassment can lead to (and has led to) fear, depression, and suicide in the worst cases.
The anonymity of the Internet partially enables online harassment, since it allows perpetrators to hide behind usernames or false images while they harass others — there is little accountability. However, 66% of online harassment happens on social media platforms, where people's names and images are usually readily available. This indicates that anonymity is not the only factor, and not even the main factor. Rather, the separation of the Internet from the physical world, that which makes it less "real," is what enables harassers to treat it as entirely different experience. They can say across a keyboard what they might never say face-to-face.
To increase my understanding of the problem, I made two three-dimensional pieces – a functioning clock and an exhibit wall. Each project explored different aspects of online harassment and implored the audience to keep compassion and kindness in mind while interacting with others digitally.
Another goal was to create a campaign which could tackle the problem on a larger, more definite scale. To learn from others' attempts, I studied two recent, real-world campaigns against online harassment, Zero Trollerance and HeartMob. Each of these received significant amounts of good press on online news outlets, but people who enjoyed or were helped by those campaigns were grossly outnumbered by those who criticized and even lambasted those campaigns, for various reasons.
I determined that the reactive nature of those campaigns was the main cause of their failure, so I created a proactive campaign with the goal of preventing online harassment, rather than correcting it. I designed the beginnings of "You & I," a multiplayer online game for children ages 4–6, which would encourage positive interaction between players through its very game mechanics. Ideally, the habits formed by the children while playing this game would carry over to their future Internet experiences, and a new generation of kinder, more cooperative, "native" Internet users would arise, reducing the amount of harassment seen on the Internet.
According to a survey performed by Pew Research in 2014, 40% of Internet users have experienced online harassment. 18% had experienced severe harassment – stalking, sexual harassment, physical threats – while 22% had only experienced less severe harassment, such as name-calling. Women ages 18–24 receive a disproportionately large percentage of all severe online harassment. The emotional trauma suffered from severe or long-term harassment can lead to (and has led to) fear, depression, and suicide in the worst cases.
The anonymity of the Internet partially enables online harassment, since it allows perpetrators to hide behind usernames or false images while they harass others — there is little accountability. However, 66% of online harassment happens on social media platforms, where people's names and images are usually readily available. This indicates that anonymity is not the only factor, and not even the main factor. Rather, the separation of the Internet from the physical world, that which makes it less "real," is what enables harassers to treat it as entirely different experience. They can say across a keyboard what they might never say face-to-face.
To increase my understanding of the problem, I made two three-dimensional pieces – a functioning clock and an exhibit wall. Each project explored different aspects of online harassment and implored the audience to keep compassion and kindness in mind while interacting with others digitally.
Another goal was to create a campaign which could tackle the problem on a larger, more definite scale. To learn from others' attempts, I studied two recent, real-world campaigns against online harassment, Zero Trollerance and HeartMob. Each of these received significant amounts of good press on online news outlets, but people who enjoyed or were helped by those campaigns were grossly outnumbered by those who criticized and even lambasted those campaigns, for various reasons.
I determined that the reactive nature of those campaigns was the main cause of their failure, so I created a proactive campaign with the goal of preventing online harassment, rather than correcting it. I designed the beginnings of "You & I," a multiplayer online game for children ages 4–6, which would encourage positive interaction between players through its very game mechanics. Ideally, the habits formed by the children while playing this game would carry over to their future Internet experiences, and a new generation of kinder, more cooperative, "native" Internet users would arise, reducing the amount of harassment seen on the Internet.
ContributorsWilliams, Peter Ross (Author) / Sanft, Alfred (Thesis director) / Heywood, William (Committee member) / The Design School (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Fire Shelter Foam Assist is meant as a firefighter's last effort of survival when a wildfire threatens their position. When deployed, it will cover the firefighter as the fire blows over. By reducing the time of deployment and simplifying the process, firefighters will have more time to ensure the area around them is cleared. The Fire Shelter Foam Assist has features that allow it to auto deploy around the firefighter through the use of fire foam retardant. The fire foam retardant inflates the shelter as well as provides an extra layer of protection against the wildfire.
ContributorsSmith, Tori Elizabeth (Author) / Shin, Dosun (Thesis director) / McDermott, Lauren (Committee member) / Barrett, The Honors College (Contributor) / Herberger Institute for Design and the Arts (Contributor) / The Design School (Contributor)
Created2015-05
Description
I set out to better understand the issues, perceptions & solutions surrounding drought. The question that compelled my project was "What might be all the ways that we can improve the experience of conserving, reusing & educating on the topic of water." Through the process of design research I developed a system of products that improves the user experiences surrounding water. The result is IOW, an intelligent 3-product system that aims to make your water needs & wants smarter & less wasteful.
ContributorsShappee, Christian Kyle (Author) / Shin, Dosun (Thesis director) / McDermott, Lauren (Committee member) / Barrett, The Honors College (Contributor) / Herberger Institute for Design and the Arts (Contributor) / School of Sustainability (Contributor) / The Design School (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
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
Engineering is a heavily male-dominated field and females are significantly less likely to choose an engineering-related major or career path. At the age of six years old, females start believing that their male peers are smarter than them, leading them to pursue less ambitious careers. The children's book Lyla B. An Engineering Legacy was created to encourage more young girls to discover their own potential and pursue engineering as a career. To explore the efficacy of the book on its target consumers, a pilot study was performed with first and second grade children. The participants' engineering knowledge; fixed and failure mindset beliefs; STEM (Science, Technology, Engineering, and Math) interest, competency, and career aspirations; and stereotype beliefs were evaluated before and after being read the book to determine if the story has a positive impact on children. Additionally, the satisfaction of the participants towards both the book and main character were analyzed quantitatively and qualitatively. Overall, the results of the study suggest that the book has a positive impact on the interest and competency of STEM fields and the stereotype beliefs that the children had towards engineers. The study also suggests that the book decreases fixed and failure mindsets and that the participants were satisfied with the overall concept of the book and main character, Lyla.
ContributorsPiatak, Catherine (Co-author) / Seelhammer, Marissa Leigh (Co-author) / Torrence, Kelly (Co-author) / Miller, Cindy (Thesis director) / Jordan, Shawn (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
One of the primary bottlenecks to chemical production in biological organisms is the toxicity of the chemical. Overexpression of efflux pumps has been shown to increase tolerance to aromatic compounds such as styrene and styrene oxide. Tight control of pump expression is necessary to maximize titers and prevent excessive strain on the cells. This study aimed to identify aromatic-sensitive native promoters and heterologous biosensors for construction of closed-loop control of efflux pump expression in E. coli. Using a promoter library constructed by Zaslaver et al., activation was measured through GFP output. Promoters were evaluated for their sensitivity to the addition of one of four aromatic compounds, their "leaking" of signal, and their induction threshold. Out of 43 targeted promoters, 4 promoters (cmr, mdtG, yahN, yajR) for styrene oxide, 2 promoters (mdtG, yahN) for styrene, 0 promoters for 2-phenylethanol, and 1 promoter for phenol (pheP) were identified as ideal control elements in aromatic bioproduction. In addition, a series of three biosensors (NahR, XylS, DmpR) known to be inducible by other aromatics were screened against styrene oxide, 2-phenylethanol, and phenol. The targeted application of these biosensors is aromatic-induced activation of linked efflux pumps. All three biosensors responded strongly in the presence of styrene oxide and 2-phenylethanol, with minor activation in the presence of phenol. Bioproduction of aromatics continues to gain traction in the biotechnology industry, and the continued discovery of aromatic-inducible elements will be essential to effective pathway control.
ContributorsXu, Jimmy (Author) / Nielsen, David (Thesis director) / Wang, Xuan (Committee member) / School of Life Sciences (Contributor) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Rebuilt is a project that looks to understand what Syrian refugees experience in camps, specifically Za'atari, the world's largest Syrian camp. The intent of Rebuilt was to create a product that would help their living conditions. By applying Design Thinking & Process, Rebuilt ultimately yielded a room partition system to help improve the living conditions of refugees. To design a product for a world most of the world is ignorant of, research is paramount. Research for Rebuilt involved gather many facts from various international databases, such as UNHCR and Mercy Corps. By understanding the demographics, the culture, and needs, Rebuilt was able to focus on some key points that lead to a potential design project: over half of the camp is consisted of adolescents (under age 18), and are living in small, essentially shipping-container homes, and the environment of the Jordanian desert where the camp is situated is extremely variable between freezing winters and blistering summers. Looking over the resources provided by humanitarian organizations, Rebuilt pinpointed a missing niche product that could help the living conditions of refugee's lives: a room partition system that could regulate ambient temperatures. The need for private space is important for the development of a refugee adolescent as it encourages stability and a sense of home. Ambient temperature is also vastly important for the productivity and health of anyone. Rebuilt is consisted of two main parts: the design of a bracket that could be used to accommodate the widths of multiple building materials and would be cheap to manufacture, and a pre-made panel that incorporated the use of phase-change-material technology. The design process is documented with a finalized design that should be low-cost and light-weight to ship from manufacturers to those in need.
ContributorsLee, Anna Jade (Author) / Shin, Dosun (Thesis director) / Bacalzo, Dean (Committee member) / Department of Marketing (Contributor) / The Design School (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05