Matching Items (27)
Filtering by

Clear all filters

156042-Thumbnail Image.png
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

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.
ContributorsDaer, René (Author) / Haynes, Karmella (Thesis advisor) / Brafman, David (Committee member) / Nielsen, David (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2017
156623-Thumbnail Image.png
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,

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
157062-Thumbnail Image.png
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

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
131525-Thumbnail Image.png
Description
The original version of Helix, the one I pitched when first deciding to make a video game
for my thesis, is an action-platformer, with the intent of metroidvania-style progression
and an interconnected world map.

The current version of Helix is a turn based role-playing game, with the intent of roguelike
gameplay and a dark

The original version of Helix, the one I pitched when first deciding to make a video game
for my thesis, is an action-platformer, with the intent of metroidvania-style progression
and an interconnected world map.

The current version of Helix is a turn based role-playing game, with the intent of roguelike
gameplay and a dark fantasy theme. We will first be exploring the challenges that came
with programming my own game - not quite from scratch, but also without a prebuilt
engine - then transition into game design and how Helix has evolved from its original form
to what we see today.
ContributorsDiscipulo, Isaiah K (Author) / Meuth, Ryan (Thesis director) / Kobayashi, Yoshihiro (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
136549-Thumbnail Image.png
Description
A primary goal in computer science is to develop autonomous systems. Usually, we provide computers with tasks and rules for completing those tasks, but what if we could extend this type of system to physical technology as well? In the field of programmable matter, researchers are tasked with developing synthetic

A primary goal in computer science is to develop autonomous systems. Usually, we provide computers with tasks and rules for completing those tasks, but what if we could extend this type of system to physical technology as well? In the field of programmable matter, researchers are tasked with developing synthetic materials that can change their physical properties \u2014 such as color, density, and even shape \u2014 based on predefined rules or continuous, autonomous collection of input. In this research, we are most interested in particles that can perform computations, bond with other particles, and move. In this paper, we provide a theoretical particle model that can be used to simulate the performance of such physical particle systems, as well as an algorithm to perform expansion, wherein these particles can be used to enclose spaces or even objects.
ContributorsLaff, Miles (Author) / Richa, Andrea (Thesis director) / Bazzi, Rida (Committee member) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor)
Created2015-05
136691-Thumbnail Image.png
Description
Covering subsequences with sets of permutations arises in many applications, including event-sequence testing. Given a set of subsequences to cover, one is often interested in knowing the fewest number of permutations required to cover each subsequence, and in finding an explicit construction of such a set of permutations that has

Covering subsequences with sets of permutations arises in many applications, including event-sequence testing. Given a set of subsequences to cover, one is often interested in knowing the fewest number of permutations required to cover each subsequence, and in finding an explicit construction of such a set of permutations that has size close to or equal to the minimum possible. The construction of such permutation coverings has proven to be computationally difficult. While many examples for permutations of small length have been found, and strong asymptotic behavior is known, there are few explicit constructions for permutations of intermediate lengths. Most of these are generated from scratch using greedy algorithms. We explore a different approach here. Starting with a set of permutations with the desired coverage properties, we compute local changes to individual permutations that retain the total coverage of the set. By choosing these local changes so as to make one permutation less "essential" in maintaining the coverage of the set, our method attempts to make a permutation completely non-essential, so it can be removed without sacrificing total coverage. We develop a post-optimization method to do this and present results on sequence covering arrays and other types of permutation covering problems demonstrating that it is surprisingly effective.
ContributorsMurray, Patrick Charles (Author) / Colbourn, Charles (Thesis director) / Czygrinow, Andrzej (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Physics (Contributor)
Created2014-12
136516-Thumbnail Image.png
Description
Bots tamper with social media networks by artificially inflating the popularity of certain topics. In this paper, we define what a bot is, we detail different motivations for bots, we describe previous work in bot detection and observation, and then we perform bot detection of our own. For our bot

Bots tamper with social media networks by artificially inflating the popularity of certain topics. In this paper, we define what a bot is, we detail different motivations for bots, we describe previous work in bot detection and observation, and then we perform bot detection of our own. For our bot detection, we are interested in bots on Twitter that tweet Arabic extremist-like phrases. A testing dataset is collected using the honeypot method, and five different heuristics are measured for their effectiveness in detecting bots. The model underperformed, but we have laid the ground-work for a vastly untapped focus on bot detection: extremist ideal diffusion through bots.
ContributorsKarlsrud, Mark C. (Author) / Liu, Huan (Thesis director) / Morstatter, Fred (Committee member) / Barrett, The Honors College (Contributor) / Computing and Informatics Program (Contributor) / Computer Science and Engineering Program (Contributor) / School of Mathematical and Statistical Sciences (Contributor)
Created2015-05
136265-Thumbnail Image.png
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

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
136133-Thumbnail Image.png
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

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
137224-Thumbnail Image.png
Description
The ability to edit chromosomal regions is an important tool for the study of gene function and the ability to engineer synthetic gene networks. CRISPR-Cas systems, a bacterial RNA-guided immune system against foreign nucleic acids, have recently been engineered for a plethora of genome engineering and transcriptional regulation applications. Here

The ability to edit chromosomal regions is an important tool for the study of gene function and the ability to engineer synthetic gene networks. CRISPR-Cas systems, a bacterial RNA-guided immune system against foreign nucleic acids, have recently been engineered for a plethora of genome engineering and transcriptional regulation applications. Here we employ engineered variants of CRISPR systems in proof-of-principle experiments demonstrating the ability of CRISPR-Cas derived single-DNA-strand cutting enzymes (nickases) to direct host-cell genomic recombination. E.coli is generally regarded as a poorly recombinogenic host with double-stranded DNA breaks being highly lethal. However, CRISPR-guided nickase systems can be easily programmed to make very precise, non-lethal, incisions in genomic regions directing both single reporter gene and larger-scale recombination events deleting up to 36 genes. Genome integrated repetitive elements of variable sizes can be employed as sites for CRISPR induced recombination. We project that single-stranded based editing methodologies can be employed alongside preexisting genome engineering techniques to assist and expedite metabolic engineering and minimalized genome research.
ContributorsStandage-Beier, Kylie S (Author) / Wang, Xiao (Thesis director) / Haynes, Karmella (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05