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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
Engineering is a heavily male-dominated field and females are significantly less likely to choose an engineering-related major or career path. At the age of six years old, females start believing that their male peers are smarter than them, leading them to pursue less ambitious careers. The children's book Lyla B. An Engineering Legacy was created to encourage more young girls to discover their own potential and pursue engineering as a career. To explore the efficacy of the book on its target consumers, a pilot study was performed with first and second grade children. The participants' engineering knowledge; fixed and failure mindset beliefs; STEM (Science, Technology, Engineering, and Math) interest, competency, and career aspirations; and stereotype beliefs were evaluated before and after being read the book to determine if the story has a positive impact on children. Additionally, the satisfaction of the participants towards both the book and main character were analyzed quantitatively and qualitatively. Overall, the results of the study suggest that the book has a positive impact on the interest and competency of STEM fields and the stereotype beliefs that the children had towards engineers. The study also suggests that the book decreases fixed and failure mindsets and that the participants were satisfied with the overall concept of the book and main character, Lyla.
ContributorsPiatak, Catherine (Co-author) / Seelhammer, Marissa Leigh (Co-author) / Torrence, Kelly (Co-author) / Miller, Cindy (Thesis director) / Jordan, Shawn (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
Description
In this creative project, our goal was to establish a student lead service organization dedicated to raising money and awareness for a selected medical issue through an interactive carnival event. In doing so, we were able to identify the potential obstacles and pathways that are required for service organizations within Arizona State University. Our experience provides a guideline for future students looking to organize charitable events on campus. This paper discusses several essential skills for running a charitable student organization, including establishing a brand, managing finances, cultivating business relationships, and marketing the cause. It is our hope that future students can learn from our experience and find success in similar endeavors.
ContributorsStoddard, Stacy Dawn (Co-author) / Wong, Brittney (Co-author) / Hultsman, Wendy (Thesis director) / Holland-Malcom, Jan (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The novella Flicker by Rachel Ponstein is a climate fiction story. It draws influence from the post-apocalyptic and dystopian genres as well as classic gothic literature. The story utilizes elements of gothic literature including Freud's Uncanny, uneven framing, and bildungsroman. It also utilizes subhuman species to incite conversation about the importance of perspective and the use of an alternative lens on the post-Reckoning world. The disaster story is ambiguous to focus the reader on the importance of the characters and their progress throughout the journey rather than the overall plotline. The analysis below serves as an explanation for the intentional decisions made to fit a sub-genre and engage the reader in an intellectual conversation about the issues broached.
ContributorsPonstein, Rachel Kay (Author) / Fette, Donald (Thesis director) / Hoyt, Heather (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The goal of this project was to create a card game that would quickly and easily allow medical professionals to learn important information. This project seeks to advance ways in which medical staff gain information about disease outbreaks through the creation of a card game which teaches players the proper steps and procedure to triage and treat patients who are suspected to have Ebola Hemorrhagic Fever, which was not done properly during the recent outbreak. To create this game, research was conducted on the information given by the Center for Disease Control and Prevention (CDC) on the various steps to triaging those who were suspected of having Ebola. Various prototypes of the game were made and tested to optimize the win-lose ratio while still being an enjoyable game to play. This card game is fast-paced, small, and can be played either individually or with more than one person. It is loosely based off of Solitaire. This game has gone through three prototypes of the cards as well as a few brief testing periods. Through the methods and procedure used in this game's creation, it has been concluded that this method is a great way to easily teach players a proper procedure, and that this method of game can be applied to other disease breakouts and even to other fields where information must be learned quickly. Future steps for this game include improving the graphic art used in the cards, and continuing on to create a smartphone application.
ContributorsHenriksen, Carissa (Co-author) / Pratt, Breanna (Co-author) / LaBelle, Jeffrey (Thesis director) / Coursen, Jerry (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
The purpose of this creative project was to establish the foundation of an educational program that teaches financial literacy to the local homeless population. The name of this program is stillHUMAN. The project consisted of two parts, a needs analysis and a prototyping phase. The needs analysis was conducted at the Phoenix Rescue Mission Center, a faith-based homeless shelter that caters to male "clients", through written surveys and one-on-one interviews. Before interviewing the clients, the team acquired IRB approval as well as consent from the Center to carry out this study. These needs were then organized into a House of Quality. We concluded from Part 1 that we would need to create 3 - 7-minute-long video modules that would be available on an online platform and covered topics including professional development, budgeting, credit, and Internet literacy. In order to commence Part 2, each team member recorded a video module. These three videos collectively conveyed instruction regarding how to write a resume, use the Internet and fill out an application online, and how to budget money. These videos were uploaded to YouTube and shown to clients at Phoenix Rescue Mission, who were each asked to fill out a feedback survey afterwards. The team plans to use these responses to improve the quality of future video modules and ultimately create a holistic lesson plan that covers all financial literacy topics the clients desire. A website was also made to store future videos. The team plans to continue with this project post-graduation. Future tasks include creating and testing the a complete lesson plan, establishing a student organization at Arizona State University and recruiting volunteers from different disciplines, and creating an on-site tutoring program so clients may receive individualized attention. Once the lesson plan is demonstrated to be effective at Phoenix Rescue Mission, we plan to administer this lesson plan at other local homeless shelters and assess its efficacy in a non-faithbased and non-male environment. After a successful financial literacy program has been created, we aim to create lesson plans for other topics, including health literacy, human rights, and basic education. Ultimately stillHUMAN will become a sustainable program that unites the efforts of students and professionals to improve the quality of life of the homeless population.
ContributorsKim, Michael (Co-author) / Gulati, Guneet (Co-author) / Vanood, Aimen (Co-author) / Ganesh, Tirupalavanam (Thesis director) / Shrake, Scott (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Life Sciences (Contributor) / Department of Physics (Contributor) / Department of Psychology (Contributor) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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 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
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 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.
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