Matching Items (13)
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
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
136696-Thumbnail Image.png
Description
Bacteria have been shown to possess a large array of regulatory mechanisms to not just respond to a diverse array of environmental stresses, but to injurious artificial proteins as well. A previous investigation introduced DX, a man-made ATP sequestering protein into Escherichia coli (E. coli) which resulted in the formation

Bacteria have been shown to possess a large array of regulatory mechanisms to not just respond to a diverse array of environmental stresses, but to injurious artificial proteins as well. A previous investigation introduced DX, a man-made ATP sequestering protein into Escherichia coli (E. coli) which resulted in the formation of novel endoliposome structures and induced a viable but non-culturable state (VBNC) that was not easily reversed. It was hypothesized that the broadly conserved bacterial stringent response pathway may have been responsible for the observed phenotypic changes. With the goal of unveiling the molecular mechanism behind this novel response, changes in cellular morphology and physiology upon DX expression were assessed in a population of E. coli encoding a dysfunctional relA gene, one of the two genes controlling the induction of the stringent response. It was ultimately shown that RelA directly contributed to cellular filamentation, endoliposome structure formation, and the induction of a VBNC state. While the stringent response has been extensively shown to induce a VBNC state, to our knowledge, relA has not yet been shown to induce filamentation or coordinate the formation of endoliposome structures in bacteria. As the stringent response has been shown to be increasingly involved in antibiotic tolerance, this study provided an exciting opportunity to further characterize this adaptive response pathway to aid in the future development of novel therapeutics. In addition to this, this study continued to highlight that the DX protein may serve one of the first tools to allow for the direct selection of bacteria in a VBNC state by morphologically distinguishing non-culturable cells through cellular filamentation.
ContributorsFrost, Fredrick Charles (Author) / Chaput, John (Thesis director) / Wachter, Rebekka (Committee member) / Korch, Shaleen (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-12
136118-Thumbnail Image.png
Description
Nucleic acids encode the information required to create life, and polymerases are the gatekeepers charged with maintaining the storage and flow of this genetic information. Synthetic biologists utilize this universal property to modify organisms and other systems to create unique traits or improve the function of others. One of the

Nucleic acids encode the information required to create life, and polymerases are the gatekeepers charged with maintaining the storage and flow of this genetic information. Synthetic biologists utilize this universal property to modify organisms and other systems to create unique traits or improve the function of others. One of the many realms in synthetic biology involves the study of biopolymers that do not exist naturally, which is known as xenobiology. Although life depends on two biopolymers for genetic storage, it may be possible that alternative molecules (xenonucleic acids – XNAs), could be used in their place in either a living or non-living system. However, implementation of an XNA based system requires the development of polymerases that can encode and decode information stored in these artificial polymers. A strategy called directed evolution is used to modify or alter the function of a protein of interest, but identifying mutations that can modify polymerase function is made problematic by their size and overall complexity. To reduce the amount of sequence space that needs to be samples when attempting to identify polymerase variants, we can try to make informed decisions about which amino acid residues may have functional roles in catalysis. An analysis of Family B polymerases has shown that residues which are involved in substrate specificity are often highly conserved both at the sequence and structure level. In order to validate the hypothesis that a strong correlation exists between structural conservation and catalytic activity, we have selected and mutated residues in the 9°N polymerase using a loss of function mutagenesis strategy based on a computational analysis of several homologues from a diverse range of taxa. Improvement of these models will hopefully lead to quicker identification of loci which are ideal engineering targets.
ContributorsHaeberle, Tyler Matthew (Author) / Chaput, John (Thesis director) / Chen, Julian (Committee member) / Larsen, Andrew (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (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
154562-Thumbnail Image.png
Description
Synthetic biology is a novel method that reengineers functional parts of natural genes of interest to build new biomolecular devices able to express as designed. There is increasing interest in synthetic biology due to wide potential applications in various fields such as clinics and fuel production. However, there are still

Synthetic biology is a novel method that reengineers functional parts of natural genes of interest to build new biomolecular devices able to express as designed. There is increasing interest in synthetic biology due to wide potential applications in various fields such as clinics and fuel production. However, there are still many challenges in synthetic biology. For example, many natural biological processes are poorly understood, and these could be more thoroughly studied through model synthetic gene networks. Additionally, since synthetic biology applications may have numerous design constraints, more inducer systems should be developed to satisfy different requirements for genetic design.

This thesis covers two topics. First, I attempt to generate stochastic resonance (SR) in a biological system. Synthetic bistable systems were chosen because the inducer range in which they exhibit bistability can satisfy one of the three requirements of SR: a weak periodic force is unable to make the transition between states happen. I synthesized several different bistable systems, including toggle switches and self-activators, to select systems matching another requirement: the system has a clear threshold between the two energy states. Their bistability was verified and characterized. At the same time, I attempted to figure out the third requirement for SR – an effective noise serving as the stochastic force – through one of the most widespread toggles, the mutual inhibition toggle, in both yeast and E. coli. A mathematic model for SR was written and adjusted.

Secondly, I began work on designing a new genetic system capable of responding to pulsed magnetic fields. The operators responding to pulsed magnetic stimuli in the rpoH promoter were extracted and reorganized. Different versions of the rpoH promoter were generated and tested, and their varying responsiveness to magnetic fields was recorded. In order to improve efficiency and produce better operators, a directed evolution method was applied with the help of a CRISPR-dCas9 nicking system. The best performing promoters thus far show a five-fold difference in gene expression between trials with and without the magnetic field.
ContributorsHu, Hao (Author) / Wang, Xiao (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2016
135297-Thumbnail Image.png
Description
Current research into live-cell dynamics, particularly those relating to chromatin structure and remodeling, are limited. The tools that are used to detect state changes in chromatin, such as Chromatin Immunoprecipitation and qPCR, require that the cell be killed off. This limits the ability of researchers to pinpoint changes in live

Current research into live-cell dynamics, particularly those relating to chromatin structure and remodeling, are limited. The tools that are used to detect state changes in chromatin, such as Chromatin Immunoprecipitation and qPCR, require that the cell be killed off. This limits the ability of researchers to pinpoint changes in live cells over a longer period of time. As such, there is a need for a live-cell sensor that can detect chromatin state changes. The Chromometer is a transgenic chromatin state sensor designed to better understand human cell fate and the chromatin changes that occur. HOXD11.12, a DNA sequence that attracts repressive Polycomb group (PCG) proteins, was placed upstream of a core promoter-driven fluorescent reporter (AmCyan fluorescent protein, CFP) to link chromatin repression to a CFP signal. The transgene was stably inserted at an ectopic site in U2-OS (osteosarcoma) cells. Expression of CFP should reflect the epigenetic state at the HOXD locus, where several genes are regulated by Polycomb to control cell differentiation. U2-OS cells were transfected with the transgene and grown under selective pressure. Twelve colonies were identified as having integrated parts from the transgene into their genomes. PCR testing verified 2 cell lines that contain the complete transgene. Flow cytometry indicated mono-modal and bimodal populations in all transgenic cell colonies. Further research must be done to determine the effectiveness of this device as a sensor for live cell state change detection.
ContributorsBarclay, David (Co-author) / Simper, Jan (Co-author) / Haynes, Karmella (Thesis director) / Brafman, David (Committee member) / School of Life Sciences (Contributor) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
132709-Thumbnail Image.png
Description
Cell fate is a complex and dynamic process with many genetic components. It has often been likened to “multistable” mathematical systems because of the numerous possible “stable” states, or cell types, that cells may end up in. Due to its complexity, understanding the process of cell fate and

Cell fate is a complex and dynamic process with many genetic components. It has often been likened to “multistable” mathematical systems because of the numerous possible “stable” states, or cell types, that cells may end up in. Due to its complexity, understanding the process of cell fate and differentiation has proven challenging. A better understanding of cell differentiation has applications in regenerative stem cell therapies, disease pathologies, and gene regulatory networks.
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.
ContributorsLoveday, Tristan Andre (Author) / Wang, Xiao (Thesis director) / Brafman, David (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
165210-Thumbnail Image.png
Description

Industries and research utilizing genetically-engineered organisms are often subject to strict containment requirements such as physical isolation or specialized equipment to prevent an unintended escape. A relatively new field of research looks for ways to engineer intrinsic containment techniques- genetic safeguards that prevent an organism from surviving outside of specific

Industries and research utilizing genetically-engineered organisms are often subject to strict containment requirements such as physical isolation or specialized equipment to prevent an unintended escape. A relatively new field of research looks for ways to engineer intrinsic containment techniques- genetic safeguards that prevent an organism from surviving outside of specific conditions. As interest in this field has grown over the last few decades, researchers in molecular and synthetic biology have discovered many novel ways to accomplish this containment, but the current literature faces some ambiguity and overlap in the ways they describe various biocontainment methods. Additionally, the way publications report the robustness of the techniques they test is inconsistent, making it uncertain how regulators could assess the safety and efficacy of these methods if they are eventually to be used in practical, consumer applications. This project organizes and clarifies the descriptions of these techniques within an interactive flowchart, linking to definitions and references to publications on each within an Excel table. For each reference, variables such as the containment approach, testing methods, and results reported are compiled, to illustrate the varying degrees to which these techniques are tested.

ContributorsDilly, Leon (Author) / Frow, Emma (Thesis director) / Vogel, Kathleen (Committee member) / Gillum, David (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2022-05
165211-Thumbnail Image.png
Description

Industries and research utilizing genetically-engineered organisms are often subject to strict containment requirements such as physical isolation or specialized equipment to prevent an unintended escape. A relatively new field of research looks for ways to engineer intrinsic containment techniques- genetic safeguards that prevent an organism from surviving outside of specific

Industries and research utilizing genetically-engineered organisms are often subject to strict containment requirements such as physical isolation or specialized equipment to prevent an unintended escape. A relatively new field of research looks for ways to engineer intrinsic containment techniques- genetic safeguards that prevent an organism from surviving outside of specific conditions. As interest in this field has grown over the last few decades, researchers in molecular and synthetic biology have discovered many novel ways to accomplish this containment, but the current literature faces some ambiguity and overlap in the ways they describe various biocontainment methods. Additionally, the way publications report the robustness of the techniques they test is inconsistent, making it uncertain how regulators could assess the safety and efficacy of these methods if they are eventually to be used in practical, consumer applications. This project organizes and clarifies the descriptions of these techniques within an interactive flowchart, linking to definitions and references to publications on each within an Excel table. For each reference, variables such as the containment approach, testing methods, and results reported are compiled, to illustrate the varying degrees to which these techniques are tested.

ContributorsDilly, Leon (Author) / Frow, Emma (Thesis director) / Vogel, Kathleen (Committee member) / Gillum, David (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2022-05