Matching Items (3)
Filtering by

Clear all filters

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
132559-Thumbnail Image.png
Description
Oncolytic virotherapy (OV) is the use of viruses that do not target normal human cells to infect and destroy cancer cells; some also stimulate the immune system against the tumors. Myxoma virus (VMYX) is a candidate for use as an oncolytic agent, as it is not pathogenic to humans and

Oncolytic virotherapy (OV) is the use of viruses that do not target normal human cells to infect and destroy cancer cells; some also stimulate the immune system against the tumors. Myxoma virus (VMYX) is a candidate for use as an oncolytic agent, as it is not pathogenic to humans and can infect a variety of human cancer cells. VMYX also can initiate immune responses against the virus-infected tumor. Thus, we investigated the oncolytic efficacy of a few recombinant constructs of VMYX on triple-negative breast cancer (TNBC), a highly aggressive subtype of breast cancer with limited treatment options. TNBC lacks an estrogen receptor, progesterone receptor, and HER2, which render hormone-based therapies useless. Further challenges of TNBC include early metastasis and recurrence, as well as poor prognosis due to a lack of molecular targets. Here, we utilized 4T1-Luc2 cells, an in vitro mouse model of TNBC, to examine the oncolytic potential of recombinant viral constructs of VMYX. Ability to infect was measured by fluorescence intensity, while ability to induce cytotoxicity was measured through MTS and SYTOX assays. Further characterization of cell death was performed using Caspase 3/7 activity assay, immunofluorescent staining and confocal microscopy to detect ecto-expression of calreticulin, and ATP release assays. We demonstrated the ability of recombinant VMYX constructs to infect and induce cell death in 4T1-Luc2 cells. VMYX-p14-FAST-GFP induced the most cell death, while VMYX-M011LKO-GFP best activated Caspase 3/7. Through ATP release assays and examination of ecto-expression of calreticulin, preliminary data indicated that VYX-135KO-GFP is unable to stimulate immunogenic cell death, a form of cell death that stimulates an adaptive immune response, in these cells. Future directions include further characterization of cell death in vitro, as well as in vivo studies.
ContributorsBelmont, Laura (Author) / McFadden, Grant (Thesis director) / Chen, Julian (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05