Matching Items (17)
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- All Subjects: Biomedical
- All Subjects: Synthetic Biology
- Creators: Harrington Bioengineering Program
- Creators: Chaput, John C
- Member of: Theses and Dissertations
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
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
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
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
Spasticity is a neurological disorder in which a target group of muscles remain in a contracted state. In addition to interfering with the function of these muscles, spasticity causes chronic pain and discomfort. Often found in patients with cerebral palsy, multiple sclerosis, or stroke history, spasticity affects an estimated twelve million people worldwide. Not only does spasticity cause discomfort and loss of function, but the condition can lead to contractures, or permanent shortenings of the muscle and connective tissue, if left untreated. Current treatments for spasticity are primarily different forms of muscle relaxant pharmaceuticals. Almost all of these drugs, however, carry unwanted side effects, including total muscle weakness, liver toxicity, and possible dependence. Additionally, kinesiotherapy, conducted by physical therapists at rehabilitation clinics, is often prescribed to people suffering from spasticity. Since kinesiotherapy requires frequent practice to be effective, proper treatment requires constant professional care and clinic appointments, discouraging patient compliance. Consequently, a medical device that could automate relief for spasticity outside of a clinic is desired in the market. While a number of different dynamic splints for hand spasticity are currently on the market, research has shown that these devices, which simply brace the hand in an extended position, do not work through any mechanism to decrease spastic tension over time. Two methods of temporarily reducing spasticity that have been observed in clinical studies are cryotherapy, or the decrease of temperature on a target area, and electrotherapy, which is the delivery of regulated electrical pulses to a target area. It is possible that either of these mechanisms could be incorporated into a medical device aimed toward spastic relief. In fact, electrotherapy is used in a current market device called the SaeboStim, which is advertised to help stroke recovery and spastic reduction. The purpose of this paper is to evaluate the viability of a potential spastic relief device that utilizes cryotherapy to a current and closest competitor, the SaeboStim. The effectiveness of each device in relieving spasticity is reviewed. The two devices are also compared on their ability to address primary customer needs, such as convenience, ease of use, durability, and price. Overall, it is concluded that the cryotherapy device more effectively relieves hand spasticity in users, although the SaeboStim's smaller size and better convenience gives it market appeal, and reveals some of the shortcomings in the preliminary design of the cryotherapy device.
ContributorsWiedeman, Christopher Blaise (Author) / Kleim, Jeffrey (Thesis director) / Buneo, Christopher (Committee member) / W.P. Carey School of Business (Contributor) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Synthetic biology is an emerging engineering disciple, which designs and controls biological systems for creation of materials, biosensors, biocomputing, and much more. To better control and engineer these systems, modular genetic components which allow for highly specific and high dynamic range genetic regulation are necessary. Currently the field struggles to demonstrate reliable regulators which are programmable and specific, yet also allow for a high dynamic range of control. Inspired by the characteristics of the RNA toehold switch in E. coli, this project attempts utilize artificial introns and complementary trans-acting RNAs for gene regulation in a eukaryote host, S. cerevisiae. Following modification to an artificial intron, splicing control with RNA hairpins was demonstrated. Temperature shifts led to increased protein production likely due to increased splicing due to hairpin loosening. Progress is underway to demonstrate trans-acting RNA interaction to control splicing. With continued development, we hope to provide a programmable, specific, and effective means for translational gene regulation in S. cerevisae.
ContributorsDorr, Brandon Arthur (Author) / Wang, Xiao (Thesis director) / Green, Alexander (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Volume depletion can lead to migraines, dizziness, and significant decreases in a subject's ability to physically perform. A major cause of volume depletion is dehydration, or loss in fluids due to an imbalance in fluid intake to fluid excretion. Because proper levels of hydration are necessary in order to maintain both short and long term health, the ability to monitor hydration levels is growing in clinical demand. Although devices capable of monitoring hydration level exist, these devices are expensive, invasive, or inaccurate and do not offer a continuous mode of measurement. The ideal hydration monitor for consumer use needs to be characterized by its portability, affordability, and accuracy. Also, this device would need to be noninvasive and offer continuous hydration monitoring in order to accurately assess fluctuations in hydration data throughout a specified time period. One particular method for hydration monitoring that fits the majority of these criteria is known as bioelectric impedance analysis (BIA). Although current devices using BIA do not provide acceptable levels of accuracy, portability, or continuity in data collection, BIA could potentially be modified to fit many, if not all, desired customer specifications. The analysis presented here assesses the viability of using BIA as a new standard in hydration level measurement. The analysis uses data collected from 22 subjects using an existing device that employs BIA. A regression derived for estimating TBW based on the parameters of age, weight, height, sex, and impedance is presented. Using impedance data collected for each subject, a regression was also derived for estimating impedance based on the factors of age, weight, height, and sex. The derived regression was then used to calculate a new impedance value for each subject, and these new impedance values were used to estimate TBW. Through a paired-t test between the TBW values derived by using the direct measurements versus the calculated measurements of impedance, the two samples were found to be comparable. Considerations for BIA as a noninvasive measurement of hydration are discussed.
ContributorsTenorio, Jorge Antonio (Author) / LaBelle, Jeffrey (Thesis director) / Pizziconi, Vincent (Committee member) / Spano, Mark (Committee member) / Barrett, The Honors College (Contributor) / W. P. Carey School of Business (Contributor) / Harrington Bioengineering Program (Contributor)
Created2013-05
Description
The action/adventure game Grad School: HGH is the final, extended version of a BME Prototyping class project in which the goal was to produce a zombie-themed game that teaches biomedical engineering concepts. The gameplay provides fast paced, exciting, and mildly addicting rooms that the player must battle and survive through, followed by an engineering puzzle that must be solved in order to advance to the next room. The objective of this project was to introduce the core concepts of BME to prospective students, rather than attempt to teach an entire BME curriculum. Based on user testing at various phases in the project, we concluded that the gameplay was engaging enough to keep most users' interest through the educational puzzles, and the potential for expanding this project to reach an even greater audience is vast.
ContributorsNitescu, George (Co-author) / Medawar, Alexandre (Co-author) / Spano, Mark (Thesis director) / LaBelle, Jeffrey (Committee member) / Guiang, Kristoffer (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05
Description
As the rates of anxiety in adults rapidly swell, new and creative treatment methods become increasingly relevant. Individuals with an anxiety disorder may experience challenging symptoms that interfere with daily activities and impede academic and social success. The purpose of this project is to design and engineer a portable heart rate monitor that communicates with an iOS mobile application for use by individuals suffering from anxiety or panic disorders. The proposed device captures the innovation of combining biosensor feedback with new, creative therapy methods on a convenient iOS application. The device is implemented as an Arduino Uno which translates radial pulse information onto an LCD screen from a wristband. Additionally, the iOS portion uses a slow expanding and collapsing animation to guide the user through a calming breathing exercise while displaying their pulse in beats per minute. The user's awareness or his or her ability to control one's own physiological state supports and facilitates an additional form of innovative therapy. The current design of the iOS app uses a random-number generator between 40 to 125 to imitate a realistic heart rate. If the value is less than 60 or greater than 105, the number is printed in red; otherwise the heart rate is displayed in green. Future versions of this device incorporate bluetooth capabilities and potentially additional synchronous methods of therapy. The information presented in this research provides an excellent example of the integrations of new mobile technology and healthcare.
ContributorsTadayon, Ramesh (Author) / Muthuswamy, Jit (Thesis director) / Towe, Bruce (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05