Directed evolution is a powerful tool that mimics the natural selection process to engineer biomolecules with improved and altered functionalities for a wide variety of applications. The advance of biological engineering based on directed evolution techniques depends upon selection assays that can practically search large, diverse libraries for the most improved variants. In Chapter 1, we begin by discussing the potential of growth selections to serve as accessible and robust assays for directed evolution. We then delve into the existing approaches to expand the scope of targets for growth selections beyond those that are intrinsically linked to growth and the strategies implemented to improve their throughput and sensitivity.
The yeast three-hybrid (Y3H) assay is a versatile system that can expand the field of directed evolution if implemented as a growth selection for the search of large variant libraries. Although the Y3H assay has been successfully applied as a positive selection to evolve proteins with improved functions, its expansion into applications requiring a high-throughput, versatile selection against transcriptional activation has been hindered by its limited dynamic range as a counter selection. To address the limited dynamic range of the Y3H assay, we undertook a multi-pronged approach to reengineer our Y3H counter selection to have a high dynamic range. In Chapter 2, we discuss strategies to improve the dynamic range of the Y3H counter selection by maximizing the growth between cells with activated and basal reporter gene expression levels. Specifically, we elaborate on the development and characterization of two Y3H counter selections that were based on either reporter gene degradation or an alternative phototoxic reporter gene. In Chapter 3, we present our most successful strategy to improve the dynamic range of the Y3H counter selection that uses the dual tetracycline (Tet) system to increase transcriptional regulation of the reporter gene. We employed a guided strategy based on both rational design and library approaches to find the best Tet Y3H reporter gene construct with the highest dynamic range. We believe our method to engineer the best Tet Y3H reporter construct will be widely useful to synthetic biologists developing sophisticated in vivo assays that require fine-tuned reporter gene expression levels. Finally, in Chapter 4, we demonstrate the versatility of the Y3H system by developing a screen for the detection of natural product biosynthesis. This assay should have an impact for metabolic engineers that are employing directed evolution techniques to generate large metabolic pathway libraries for the overproduction of high-value small molecules in heterologous producer strains.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D8RN377K |
| Date | January 2015 |
| Creators | Harton, Marie Deborah Gaynelle |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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