This work describes the characterization of the CAN1 gene, thought to encode the arginine permease of yeast. I have identified the RNA transcript of this gene and obtained the DNA sequence, which specifies a highly hydrophobic protein with multiple potential membrane-spanning domains. I use a gene fusion approach to identify sequences within the CAN1 protein that can translocate adjacent sequences across the E.R. membrane in vitro and extend these observations by finding that the topology of E.R. insertion is conserved up to the plasma membrane in vivo. Using a series of CAN1 gene fusions to the secreted yeast killer toxin, I find that the pathway of membrane protein export to the cell surface need not be functionally distinct from that of secreted proteins. Finally, I describe a mutation in the CAN1 gene that leads to altered rates of lysine uptake and results in growth inhibition and rapid plasmid loss in the presence of lysine.

Thesis (MS)--Montana State University--Bozeman, 2009. / Typescript. Chairperson, Graduate Committee: A. Michael Babcock. Includes bibliographical references (leaves 69-103).

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Andrew D. Ellington. Vita. Includes bibliographical references. Available also from UMI company.

Thesis (Ph. D.)--Florida State University, 2004. / Advisor: Dr. Michael Blaber, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Sept. 21, 2004). Includes bibliographical references.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: expanded bed chromatography; Pichia pastoris. Includes bibliographical references (leaves 44-45).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains x, 104 p.; also includes graphics. Includes bibliographical references (p. 91-104). Available online via OhioLINK's ETD Center

Thesis (Ph. D.)--Drexel University, 2007. / Includes abstract and vita. Includes bibliographical references (leaves 108-115).

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Recent innovations and progress in machine learning algorithms from the Natural Language Processing (NLP) community have motivated efforts to apply these models and concepts to proteins. The representations generated by trained NLP models have been shown to capture important semantic and structural understanding of proteins encompassing biochemical and biophysical properties, among other key concepts. In turn, these representations have demonstrated application to protein engineering tasks including mutation analysis and design of novel proteins. Here we use this NLP paradigm in a protein engineering effort to further red shift the emission wavelength of the red fluorescent protein mNeptune684 using only a small number of functional training variants ('Low-N' scenario). The collaborative nature of this thesis with the Department of Chemistry and Biomolecular Sciences explores using these tools and methods in the rational design process.

Control of gene expression is essential for synthetic biology. Drug-inducible systems allow for gene expression levels to be precisely regulated through the administration of exogenous chemical inducers, and by combining these systems, more complex circuits with multiple inputs and outputs can be designed. However, the number of existing orthogonal drug-inducible systems is limited. Therefore, there remains a need for new, small-molecule-inducible systems that are orthogonal to both existing systems and native cellular function. Here, we focus on the development and applications of two small-molecule inducible systems for use in basic research, synthetic systems, and therapeutics: the ligand-inducible connection (LInC) system and the HaXS8-inducible system. In the LInC system, the NS3/4a protease from hepatitis C virus is used to regulate the linkage between genetically fused DNA-binding elements and transcriptional effector domains, which remain linked only in the presence of an NS3/4a protease inhibitor. The antiviral drugs which are used as inhibitors for NS3-based systems provide ideal small-molecule inputs for synthetic biology applications, as they are designed to be orthogonal to native biological function and have been tested for clinical use in humans. We demonstrate the construction and validation of a LInC transcription factor (TF) system, as well as further extension of the system with an “all-in-one” single vector design. We then continue on to describe the application of a small molecule (HaXS8) heterodimer system based on the SNAP-tag and HaloTag domains to control gene expression and protein behavior. We demonstrate the construction and validation of several HaXS8-inducible proteins, including TFs, Cre recombinase, and caspase-9. Together, our work on these two systems provides additional orthogonal methods of small molecule-inducible gene expression and protein function for use in control of cellular behavior. / 2024-05-23T00:00:00Z