Matching Items (17)
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- All Subjects: Synthetic Biology
- Creators: Harrington Bioengineering Program
- Creators: Nielsen, David
- Status: Published
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 developing countries do not have health care systems that can afford technological biomedical devices or supplies to make such devices operational. To fill this void, nonprofit organizations, like Project C.U.R.E., recondition retired biomedical instrumentation so they can send medical supplies to help these developing countries. One of the issues with this is that sometimes the devices are unusable because components or expendable supplies are not available (Bhadelia). This issue has also been shown in the Impact Evaluations that Project C.U.R.E. receives from the clinics that explain the reasons why certain devices are no longer in use. That need underlies the idea on which this honors thesis has come into being. The purpose of this honors project was to create packing lists for biomedical instruments that Project C.U.R.E. recycles. This packing list would decrease the likelihood of important items being forgotten when sending devices. If an extra fuse, battery, light bulb, cuff or transducer is the difference between a functional or a nonfunctional medical device, such a list would be of benefit to Project C.U.R.E and these developing countries. In order to make this packing list, manuals for each device were used to determine what supplies were required, what was necessary for cleaning, and what supplies were desirable but functionally optional. This list was then added into a database that could be easily navigated and could help when packing up boxes for a shipment. The database also makes adding and editing the packing list simple and easy so that as Project C.U.R.E. gets more donated devices the packing list can grow.
ContributorsGraft, Kelsey Anne (Author) / Coursen, Jerry (Thesis director) / Walters, Danielle (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
In the medical industry, there have been promising advances in the increase of new types of healthcare to the public. As of 2015, there was a 98% Premarket Approval rate, a 38% increase since 2010. In addition, there were 41 new novel drugs approved for clinical usage in 2014 where the average in the previous years from 2005-2013 was 25. However, the research process towards creating and delivering new healthcare to the public remains remarkably inefficient. It takes on average 15 years, over $900 million by one estimate, for a less than 12% success rate of discovering a novel drug for clinical usage. Medical devices do not fare much better. Between 2005-2009, there were over 700 recalls per year. In addition, it takes at minimum 3.25 years for a 510(k) exempt premarket approval. Plus, a time lag exists where it takes 17 years for only 14% of medical discoveries to be implemented clinically. Coupled with these inefficiencies, government funding for medical research has been decreasing since 2002 (2.5% of Gross Domestic Product) and is predicted to be 1.5% of Gross Domestic Product by 2019. Translational research, the conversion of bench-side discoveries to clinical usage for a simplistic definition, has been on the rise since the 1990s. This may be driving the increased premarket approvals and new novel drug approvals. At the very least, it is worth considering as translational research is directly related towards healthcare practices. In this paper, I propose to improve the outcomes of translational research in order to better deliver advancing healthcare to the public. I suggest Best Value Performance Information Procurement System (BV PIPS) should be adapted in the selection process of translational research projects to fund. BV PIPS has been shown to increase the efficiency and success rate of delivering projects and services. There has been over 17 years of research with $6.3 billion of projects and services delivered showing that BV PIPS has a 98% customer satisfaction, 90% minimized management effort, and utilizes 50% less manpower and effort. Using University of Michigan \u2014 Coulter Foundation Program's funding process as a baseline and standard in the current selection of translational research projects to fund, I offer changes to this process based on BV PIPS that may ameliorate it. As concepts implemented in this process are congruent with literature on successful translational research, it may suggest that this new model for selecting translational research projects to fund will reduce costs, increase efficiency, and increase success. This may then lead to more Premarket Approvals, more new novel drug approvals, quicker delivery time to the market, and lower recalls.
ContributorsDel Rosario, Joseph Paul (Author) / Kashiwagi, Dean (Thesis director) / Kashiwagi, Jacob (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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
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
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
As the complexity of healthcare continues to rise, the need for change in healthcare delivery is more prominent than ever. One strategy identified by the World Health Organization (WHO) for responding to these increasing complexities is the use of interprofessional practice and education to improve patient outcomes, reduce costs, and enhance the patient experience of care (Triple Aim). Interprofessional collaboration among diverse disciplines is evident on the Phoenix Biomedical Campus, integrating a wide variety of institutions and multiple health profession programs; and at the Student Health Outreach for Wellness (SHOW) free clinic, -- a successful tri-university, student-led, faculty mentored, and community-based model of interprofessional learning and care -- based in downtown Phoenix. This project conducted a comparative analysis of interprofessional components of 6 different clinical models in order to provide recommendations for best practice implementation. These models were chosen based on availability of research on interprofessionalism with their clinics. As a result, three recommendations were offered to the SHOW clinic for consideration in their efforts to improve both patient and educational outcomes. Each recommendation was intentionally formulated for its capacity to increase: interprofessionalism and collaboration between multiple disciplines pertaining to healthcare, among healthcare professionals to promote positive patient and educational outcomes. These recommendations include implementing an interprofessional education (IPE) course as a core component in an academic program's curriculum, offering faculty and professional development opportunities for faculty and mentors immersed in the interprofessional clinics, and utilization of simulation centers. Further studies will be needed to evaluate the impact these specific interventions, if adopted, on patient and educational outcomes.
ContributorsMousa, Mohammad (Co-author) / Mousa, Bakir (Co-author) / Johnson, Ross (Co-author) / Harrell, Liz (Thesis director) / Saewert, Karen (Committee member) / Harrington Bioengineering Program (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
p-Coumaric acid is used in the food, pharmaceutical, and cosmetic industries due to its versatile properties. While prevalent in nature, harvesting the compound from natural sources is inefficient, requiring large quantities of producing crops and numerous extraction and purification steps. Thus, the large-scale production of the compound is both difficult and costly. This research aims to produce p-coumarate directly from renewable and sustainable glucose using a co-culture of Yeast and E. Coli. Methods used in this study include: designing optimal media for mixed-species microbial growth, genetically engineering both strains to build the production pathway with maximum yield, and analyzing the presence of p-Coumarate and its pathway intermediates using High Performance Liquid Chromatography (HPLC). To date, the results of this project include successful integration of C4H activity into the yeast strain BY4741 ∆FDC1, yielding a strain that completely consumed trans-cinnamate (initial concentration of 50 mg/L) and produced ~56 mg/L p-coumarate, a resting cell assay of the co-culture that produced 0.23 mM p-coumarate from an initial L-Phenylalanine concentration of 1.14 mM, and toxicity tests that confirmed the toxicity of trans-cinnamate to yeast for concentrations above ~50 mg/L. The hope for this project is to create a feasible method for producing p-Coumarate sustainably.
ContributorsJohnson, Kaleigh Lynnae (Author) / Nielsen, David (Thesis director) / Thompson, Brian (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12