Synthetic biology is a nascent discipline that aims to design and construct new biological systems beyond those found in nature, ultimately using them to probe, control, or even replace existing biological systems. The success of synthetic biology depends on the assembly of a set of well-defined and modular tools. These tools should ideally be mutually compatible, reusable in different contexts, and have minimum crosstalk with endogenous proteins of the subject. The tobacco etch virus protease (TEV protease, TEVp) is a suitable candidate for such a tool due to its unique substrate specificity and high efficiency. Importantly, TEVp is capable of imitating proteolysis, a ubiquitous mechanism in nature for post-translational modifications and signal propagation. Here, TEVp is employed as a self-contained proteolytic device capable of executing biological tasks that are otherwise governed by endogenous proteins and processes. Consequently, the goal of using TEVp for synthetic manipulation of biosystems is achieved. First, a single-vector multiple gene expression strategy utilizing TEVp self-cleavage was created. This approach was used for the robust expression of up to three genes in both bacterial and mammalian cells with consistent stoichiometry. The products can then be individually purified or targeted to distinct subcellular compartments respectively. Second, a temperature-inducible TEVp was created by incremental truncation of TEVp. The 18th truncation of TEVp (tsTEVp) resulted in negligible activity at 37 °C, but retained sufficient activity at 30 °C for rapid processing of its substrates in several mammalian cell cultures. Finally, tsTEVp was applied in the context of other synthetic modules to generate a variety of biological responses. Its versatility was demonstrated as cellular processes including protein localization, cellular blebbing, protein degradation, and cell death were rewired to respond to the physical stimulus of temperature.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/43517 |
| Date | 08 January 2014 |
| Creators | Chen, Xi |
| Contributors | Truong, Kevin |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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