published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Enzyme engineering relies on changes in the amino acid sequence of an enzyme to give rise to improvements in catalytic activity, substrate specificity, thermostability, and enantioselectivity. However, beneficial amino acid substitutions in proteins are difficult to rationally predict. Large numbers of enzyme variants containing random amino acid substitutions are screened in a high throughput manner to isolate improved enzymes. Identifying improved enzymes from the resulting library of randomized variants is a current challenge in protein engineering. This work focuses on the development of high-throughput screens for a class of enzymes called hydrolases, and in particular, proteases and esterases. In the first part of this work, we have developed an assay for detecting protease activity in the cytoplasm of Escherichia coli by exploiting the SsrA protein degradation pathway and flow cytometry. In this method, a protease-cleavable linker is inserted between a fusion protein consisting of GFP and the SsrA degradation tag. The SsrA-tagged fusion protein is degraded in the cell unless a co-expressed protease cleaves the linker conferring higher cellular fluorescence. The assay can detect specific cleavage of substrates by TEV protease and human caspase-8. To apply the screen for protease engineering, we sought to evolve a TEV protease variant that has altered P1 specificity. However, in screening enzyme libraries, the clones we recovered were found to be false positives in that they did not express protease variants with the requisite specificities. These experiments provided valuable information on physiological and chemical parameters that can be employed to optimize the screen for directed evolution of novel protease activities. In the second part of this work, single bacterial cells, expressing an esterase in the periplasm, were compartmentalized in aqueous droplets of a water-in-oil emulsion also containing a fluorogenic ester substrate. The primary water-in-oil emulsion was then re-emulsified to form a water-in-oil-in-water double emulsion which was capable of being analyzed and sorted by flow cytometry. This method was used to enrich cells expressing an esterase with activity towards fluorescein dibutyrate from an excess of cells expressing an esterase with no activity. A 50-fold enrichment was achieved in one round of sorting, demonstrating the potential of this method for use as a high-throughput screen for esterase activity. This method is suitable for engineering esterases with novel catalytic specificities or higher stabilit / text

<p>Automated analysis techniques for flow cytometry data can address many of the limitations of manual analysis by providing an objective approach for the identification of cellular subsets. While automated analysis has the potential to significantly improve automated analysis, challenges remain for automated methods in cross sample analysis for large scale studies. This thesis presents new methods for data normalization, sample enrichment for rare events of interest, and cell subset relabeling. These methods build upon and extend the use of Gaussian mixture models in automated flow cytometry analysis to enable practical large scale cell subset identification.</p> / Dissertation

The content in the dissertation is divided into two main categories: (1) micro-particle characterization techniques based on elastic light scattering, and (2) ultra-compact on-chip plasmonic light concentration and its applications. For category (1), I developed two techniques, one is in vitro and the other is in the scenario of flow cytometry. I investigated theoretically and experimentally the spectra of scattered light from spherical dielectric particles at certain fixed angles, and demonstrate the linearity between the peak positions in the Fourier domain and the diameter of the particle. Based on this discovery, I demonstrate an efficient and accurate technique for in-vitro micro-particle sizing. Moreover, I theoretically analyzed the far-field elastic scattering signals from micro-particles passing through a flow cytometer with tightly focused incident beams, and established an algorithm to extract size information from the detected signals with higher accuracy than that in conventional flow cytometry systems. For category (2), I proposed an on-chip plasmonic nanofocusing technique whose unit device is a plasmonic triangle-shaped nanotaper mounted upon a dielectric optical waveguide. This structure provides highly efficient and robust light concentration into the tip of the nanotaper. Near-field measurements were performed to thoroughly investigate a fabricated sample and prove the concept. I also proposed theoretically a novel concept named phase-induced local-field configuration with logic behaviors, whose actuators are composite devices built on units of single on-chip plasmonic light concentrators mentioned above.

Thesis (M.Sc.)--University of Wollongong, 2004. / Typescript. Includes bibliographical references: leaf 115-122.

Thesis (Ph. D. in Chemistry)--Vanderbilt University, Dec. 2008. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Sept. 16, 2008). Includes bibliographical references (p. 208-224).

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Li Shi. Vita. Includes bibliographical references.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Environmental Health Sciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [109]-122).