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- Creators: School of Life Sciences
- Creators: Kiani, Samira
- Member of: Theses and Dissertations
- Resource Type: Text
- Status: Published
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
Photosynthesis is the process by which plants, algae, and bacteria use light energy to synthesize organic compounds to use as energy. Among these organisms are a kind of purple photosynthetic bacteria called Rhodobacter sphaeroides, a non-sulfur purple bacteria that grows aerobically in the dark by respiration. There have been many contributions throughout the history of this group of bacteria. Rhodobacter sphaeroides is metabolically very diverse as it has many different ways to obtain energy--aerobic respiration and anoxygenic photosynthesis being just a couple of the ways to do so. This project is part of a larger ongoing project to study different mutant strains of Rhodobacter and the different ways in which carries out electron transfer/photosynthesis. This thesis focused on the improvements made to protocol (standard procedure of site directed mutagenesis) through a more efficient technique known as infusion.
ContributorsNucuta, Diana Ileana (Author) / Woodbury, Neal (Thesis director) / Lin, Su (Committee member) / Loskutov, Andrey (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Malignant Pleural Mesothelioma is a type of lung cancer usually discovered at an advanced stage at which point there is no cure. Six primary MPM cell lines were used to conduct in vitro research to make conclusions about specific gene mutations associated with Mesothelioma. DNA exome sequencing, a time efficient and inexpensive technique, was used for identifying specific DNA mutations. Computational analysis of exome sequencing data was used to make conclusions about copy number variation among common MPM genes. Results show a CDKN2A gene heterozygous deletion in Meso24 cell line. This data is validated by a previous CRISPR-Cas9 outgrowth screen for Meso24 where the knocked-out gene caused increased Meso24 growth.
ContributorsKrdi, Ghena (Author) / Plaisier, Christopher (Thesis director) / Wilson, Melissa (Committee member) / School of Life Sciences (Contributor) / Hugh Downs School of Human Communication (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Conservatism is intrinsic to safety of emerging biotechnologies. Fear of unintended consequences, misuse, and bioterror are rightfully essential in our discussions of novel innovations. Clustered regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated proteins are no exception. This review will characterize environmental and health-related risks of CRISPR-applications and expound upon mechanisms that are or can be used to minimize risk. CRISPR is broadening access and simplifying genomic and transcriptomic editing leading to wide-range usage in all of biology. Utilization in gene therapies, gene drives, and agriculture could all be universally impactful applications that need their own safety technologies and guidelines. The initial ethical guidelines and recommendations, that will guide these technologies, are being steadily developed. However, technical advances are required to facilitate safe usage. Since the advent of CRISPR gene editing in 2012 advances to limit off-target edits (both cellular and genomic) have been developed. Delivery systems that use viral or nanoparticle packaging incorporate safety mechanisms to guard against undesirable side effects are being produced and rigorously tested. Besides its applications in basic biology and potential as a gene therapy, CRISPR had humbler beginnings. Industrially it was, albeit unknowingly, used to fend off infection in productions of yogurt batches. This was one of the earliest applications of CRISPR, however with the knowledge we now have ecological and industrial uses of CRISPR have multiplied. Gene drives have the power to spread genetic mutations throughout populations and agricultural uses to better crop genomes are also of interest. These uses have struck a chord with interest groups (environmentalists, anti-GMO groups, etc) who imagine how this technology can drastically alter species with unforeseen evolutionary changes that could reshape present-day ecosystems. This review will describe existing technologies that will safeguard humanity and its interests while fully employing CRISPRs far-reaching potentiality.
ContributorsPineda, Michael (Author) / Kiani, Samira (Thesis director) / Ebrahimkhani, Mo (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Neuroinflammation contributes significantly to the pathogenesis of Alzheimer’s and Parkinson’s diseases. However, the inflammatory pathways contributing to neurodegeneration are not well understood. Moreover, there is a need to identify changes in inflammatory signaling that may occur early in disease progression to identify potential targets for therapeutic intervention. An important step towards addressing this need is understanding how the extracellular vesicles (EVs) released by microglia can be detected in the periphery. For microglia, phagocytic macrophages, and CD 14+ monocytes share many genes and membrane- bound proteins, and there is currently no method to distinguish microglia EVs from those generated by macrophages or monocytes. Therefore, this study aims to identify membrane-bound proteins unique to microglia EVs to enable their reliable isolation. Liquid-chromatography tandem mass spectrometry analysis was used to detect proteins in the EVs from both normal and disease-associated human stem-cell differentiated microglia (iMGL), and human induced pluripotent stem cell-derived CD 14+ monocytes and macrophages. We identified 23 proteins unique to the microglial EVs, eight of which localize to the membrane and may be potential targets for isolation. This investigation also used RNA sequencing to gain insight into the contents of DAM-like and control iMGL EVs and of microglia and white blood cells in Alzheimer’s disease. We propose that the contents of microglial EVs isolated from peripheral compartments will provide crucial insight for understanding the current inflammatory state of CNS microglia. This approach could provide a means to track changes in microglial activation over time, which is critical for understanding the progression of neuroinflammatory diseases like Alzheimer's and Parkinson's. Additionally, it may offer insights into potential therapeutic targets for modulating neuroinflammation.
ContributorsLopatin, Ulia (Author) / Mastroeni, Diego (Thesis director) / Velazquez, Ramon (Committee member) / Van Keuren-Jensen, Kendall (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2024-05
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
The CRISPR/Cas9 gene-editing tool is currently in clinical trials as the excitement about its therapeutic potential is exponentially growing. However, many of the developed CRISPR based genome engineering methods cannot be broadly translated in clinical settings due to their unintended consequences. These consequences, such as immune reactions to CRISPR, immunogenic adverse events following receiving of adeno-associated virus (AAV) as one of the clinically relevant delivery agents, and CRISPR off-target activity in the genome, reinforces the necessity for improving the safety of CRISPR and the gene therapy vehicles. Research into designing more advanced CRISPR systems will allow for the increased ability of editing efficiency and safety for human applications. This work 1- develops strategies for decreasing the immunogenicity of CRISPR/Cas9 system components and improving the safety of CRISPR-based gene therapies for human subjects, 2- demonstrates the utility of this system in vivo for transient repression of components of innate and adaptive immunity, and 3- examines an inducible all-in-one CRISPR-based control switch to pave the way for controllable CRISPR-based therapies.
ContributorsMoghadam, Farzaneh (Author) / Kiani, Samira (Thesis advisor) / LaBaer, Josh (Committee member) / Ebrahimkhani, Mo (Committee member) / Arizona State University (Publisher)
Created2020
Description
Extracellular Vesicles (EVs), particularly exosomes, are of considerable interest as tumor biomarkers since tumor-derived EVs contain a broad array of information about tumor pathophysiology including its metabolic and metastatic status. However, current EV based assays cannot distinguish between EV biomarker changes by altered secretion of EVs during diseased conditions like cancer, inflammation, etc. that express a constant level of a given biomarker, stable secretion of EVs with altered biomarker expression, or a combination of these two factors. This issue was addressed by developing a nanoparticle and dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker level(s) to EV abundance, revealing average expression levels of EV biomarker under observation. In this approach, EVs are captured from complex samples (e.g. serum), stained with a lipophilic dye and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels. EVs from malignant (PANC-1) and nonmalignant pancreatic cell lines (HPNE) exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM, and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its flexible design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple swapping of the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker utilizing a workflow that is suitable for rapid clinical translation.
ContributorsRodrigues, Meryl (Author) / Hu, Tony (Thesis advisor) / Nikkhah, Mehdi (Committee member) / Kiani, Samira (Committee member) / Smith, Barbara (Committee member) / Han, Haiyong (Committee member) / Arizona State University (Publisher)
Created2019