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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

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
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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
Description
Based upon the idea of a "science fiction prototype" as originally designed by Brian David Johnson, Salomon’s House is a science fiction novella, written to be as scientifically accurate as possible and to present a balanced account of the potential social consequences of genetic engineering. It aims to explore the

Based upon the idea of a "science fiction prototype" as originally designed by Brian David Johnson, Salomon’s House is a science fiction novella, written to be as scientifically accurate as possible and to present a balanced account of the potential social consequences of genetic engineering. It aims to explore the answers to some core questions that have plagued scientists and philosophers alike while entertaining its readers with a punchy, character-driven narrative.
ContributorsMansfield, Izaac (Author) / Finn, Ed (Thesis director) / Frow, Emma (Committee member) / Barrett, The Honors College (Contributor) / Department of Information Systems (Contributor) / School for the Future of Innovation in Society (Contributor)
Created2023-05
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) / School of Earth and Space Exploration (Contributor)
Created2022-05
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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
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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
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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