Matching Items (23)
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- All Subjects: Biomaterials
- All Subjects: Synthetic Biology
- Creators: Harrington Bioengineering Program
- Creators: Brafman, 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
Myocardial infarction (MI) remains the leading cause of mortality and morbidity in the U.S., accounting for nearly 140,000 deaths per year. Heart transplantation and implantation of mechanical assist devices are the options of last resort for intractable heart failure, but these are limited by lack of organ donors and potential surgical complications. In this regard, there is an urgent need for developing new effective therapeutic strategies to induce regeneration and restore the loss contractility of infarcted myocardium. Over the past decades, regenerative medicine has emerged as a promising strategy to develop scaffold-free cell therapies and scaffold-based cardiac patches as potential approaches for MI treatment. Despite the progress, there are still critical shortcomings associated with these approaches regarding low cell retention, lack of global cardiomyocytes (CMs) synchronicity, as well as poor maturation and engraftment of the transplanted cells within the native myocardium. The overarching objective of this dissertation was to develop two classes of nanoengineered cardiac patches and scaffold-free microtissues with superior electrical, structural, and biological characteristics to address the limitations of previously developed tissue models. An integrated strategy, based on micro- and nanoscale technologies, was utilized to fabricate the proposed tissue models using functionalized gold nanomaterials (GNMs). Furthermore, comprehensive mechanistic studies were carried out to assess the influence of conductive GNMs on the electrophysiology and maturity of the engineered cardiac tissues. Specifically, the role of mechanical stiffness and nano-scale topographies of the scaffold, due to the incorporation of GNMs, on cardiac cells phenotype, contractility, and excitability were dissected from the scaffold’s electrical conductivity. In addition, the influence of GNMs on conduction velocity of CMs was investigated in both coupled and uncoupled gap junctions using microelectrode array technology. Overall, the key contributions of this work were to generate new classes of electrically conductive cardiac patches and scaffold-free microtissues and to mechanistically investigate the influence of conductive GNMs on maturation and electrophysiology of the engineered tissues.
ContributorsNavaei, Ali (Author) / Nikkhah, Mehdi (Thesis advisor) / Brafman, David (Committee member) / Migrino, Raymond Q. (Committee member) / Stabenfeldt, Sarah (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2018
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 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
Description
Traumatic brain injury (TBI) may result in numerous pathologies that cannot currently be mitigated by clinical interventions. Stem cell therapies are widely researched to address TBI-related pathologies with limited success in pre-clinical models due to limitations in transplant survival rates. To address this issue, the use of tissue engineered scaffolds as a delivery mechanism has been explored to improve survival and engraftment rates. Previous work with hyaluronic acid \u2014 laminin (HA-Lm) gels found high viability and engraftment rates of mouse fetal derived neural progenitor/stem cells (NPSCs) cultured on the gel. Furthermore, NPSCs exposed to the HA-Lm gels exhibit increased expression of CXCR4, a critical surface receptor that promotes cell migration. We hypothesized that culturing hNPCs on the HA-Lm gel would increase CXCR4 expression, and thus enhance their ability to migrate into sites of tissue damage. In order to test this hypothesis, we designed gel scaffolds with mechanical properties that were optimized to match that of the natural extracellular matrix. A live/dead assay showed that hNPCs preferred the gel with this optimized formulation, compared to a stiffer gel that was used in the CXCR4 expression experiment. We found that there may be increased CXCR4 expression of hNPCs plated on the HA-Lm gel after 24 hours, indicating that HA-Lm gels may provide a valuable scaffold to support viability and migration of hNPCs to the injury site. Future studies aimed at verifying increased CXCR4 expression of hNPCs cultured on HA-Lm gels are necessary to determine if HA-Lm gels can provide a beneficial scaffold for stem cell engraftment therapy for treating TBI.
ContributorsHemphill, Kathryn Elizabeth (Author) / Stabenfeldt, Sarah (Thesis director) / Brafman, David (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Cardiac tissue engineering is an emerging field that has the potential to regenerate and repair damaged cardiac tissues after myocardial infarction. Numerous studies have introduced hydrogel-based cardiac tissue constructs featuring suitable microenvironments for cell growth along with precise surface topographies for directed cell organization. Despite significant progress, previously developed cardiac tissue constructs have suffered from electrically insulated matrices and low cell retention. To address these drawbacks, we fabricated micropatterned hybrid hydrogel constructs (uniaxial microgrooves with 50 µm with) using a photocrosslinkable gelatin methacrylate (GelMA) hydrogel incorporated with gold nanorods (GNRs). The electrical impedance results revealed a lower impedance in the GelMA-GNR constructs versus the pure GelMA constructs. Superior electrical conductivity of GelMA-GNR hydrogels (due to incorporation of GNRs) enabled the hybrid tissue constructs to be externally stimulated using a pulse generator. Furthermore, GelMA-GNR tissue hydrogels were tested to investigate the biological characteristics of cultured cardiomyocytes. The F-actin fiber analysis results (area coverage and alignment indices) revealed higher directed (uniaxial) cytoskeleton organization of cardiac cells cultured on the GelMA-GNR hydrogel constructs in comparison to pure GelMA. Considerable increase in the coverage area of cardiac-specific markers (sarcomeric α-actinin and connexin 43) were observed on the GelMA-GNR hybrid constructs compared to pure GelMA hydrogels. Despite substantial dissimilarities in cell organization, both pure GelMA and hybrid GelMA-GNR hydrogel constructs provided a suitable microenvironment for synchronous beating of cardiomyocytes.
ContributorsMoore, Nathan Allen (Author) / Nikkhah, Mehdi (Thesis director) / Smith, Barbara (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Cancer is one of the leading causes of death globally according to the World Health Organization. Although improved treatments and early diagnoses have reduced cancer related mortalities, metastatic disease remains a major clinical challenge. The local tumor microenvironment plays a significant role in cancer metastasis, where tumor cells respond and adapt to a plethora of biochemical and biophysical signals from stromal cells and extracellular matrix (ECM) proteins. Due to these complexities, there is a critical need to understand molecular mechanisms underlying cancer metastasis to facilitate the discovery of more effective therapies. In the past few years, the integration of advanced biomaterials and microengineering approaches has initiated the development of innovative platform technologies for cancer research. These technologies enable the creation of biomimetic in vitro models with physiologically relevant (i.e. in vivo-like) characteristics to conduct studies ranging from fundamental cancer biology to high-throughput drug screening. In this review article, we discuss the biological significance of each step of the metastatic cascade and provide a broad overview on recent progress to recapitulate these stages using advanced biomaterials and microengineered technologies. In each section, we will highlight the advantages and shortcomings of each approach and provide our perspectives on future directions.
ContributorsPeela, Nitish (Author) / Nikkhah, Mehdi (Thesis director) / LaBaer, Joshua (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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
With an increased demand for more enzyme-sensitive, bioresorbable and more biodegradable polymers, various studies of copolymers have been developed. Polymers are widely used in various applications of biomedical engineering such as in tissue engineering, drug delivery and wound healing. Depending on the conditions in which polymers are used, they are modified to accommodate a specific need. For instance, polymers used in drug delivery are more efficient if they are biodegradable. This ensures that the delivery system does not remain in the body after releasing the drug. It is therefore crucial that the polymer used in the drug system possess biodegradable properties. Such modification can be done in different ways including the use of peptides to make copolymers that will degrade in the presence of enzymes. In this work, we studied the effect of a polypeptide GAPGLL on the polymer NIPAAm and compare with the previously studied Poly(NIPAAm-co-GAPGLF). Both copolymers Poly(NIPAAm-co-GAPGLL) were first synthesized from Poly(NIPAAm-co-NASI) through nucleophilic substitution by the two peptides. The synthesis of these copolymers was confirmed by 1H NMR spectra and through cloud point measurement, the corresponding LCST was determined. Both copolymers were degraded by collagenase enzyme at 25 ° C and their 1H NMR spectra confirmed this process. Both copolymers were cleaved by collagenase, leading to an increase in solubility which yielded a higher LCST compared to before enzyme degradation. Future studies will focus on evaluating other peptides and also using other techniques such as Differential Scanning Microcalorimetry (DSC) to better observe the LCST behavior. Moreover, enzyme kinetics studies is also crucial to evaluate how fast the enzyme degrades each of the copolymers.
ContributorsUwiringiyimana, Mahoro Marie Chantal (Author) / Vernon, Brent (Thesis director) / Nikkhah, Mehdi (Committee member) / 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