No description available.

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.

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.

No description available.

DNA polymerases are found in a diverse range of organisms, prokaryotes, eukaryotes, viruses and bacteriophage. T7 DNA polymerase is a replicative enzyme from E. coli bacteriophage T7. It relies on the thioredoxin binding domain (TBD) of phage gene 5 protein (gp5) and E. coli thioredoxin (Trx) for processive replication of phage DNA. Although T7 DNA polymerase is processive, it is also thermolabile. In order to design a thermostable and processive DNA polymerase, the structural stabilities of the TBD and Trx were studied in respect to their binding affinity and affect on enzyme processivity. An artificial operon was designed for coexpression of subunits of T7 DNA polymerase. By means of a 9??His-tag at the amino terminus of gp5, T7 DNA polymerase complex was purified by one-step nickel-agarose chromatography, with subunits gp5 and Trx co-eluting in a one to one molar ratio. Purified T7 DNA polymerase was assayed for polymerase activity, processivity and residual activity and compared to the commercial T7 DNA polymerase. The two enzymes were not identical with commercial T7 DNA polymerase being less processive at 37??C. Mass spectrometry of the two enzymes identified a mutation of Phe102 to Ser in the Trx subunit (TrxS102) of commercial T7 DNA polymerase. The Ser102 mutation, was found near the carboxyl terminal helix of Trx. TrxS102 was less stable than wild type Trx. In the study of the TBD structural stability, a hybrid polymerase was constructed by inserting the TBD motif into the homologous position in the Stoffel fragment of Taq DNA polymerase. The hybrid enzyme was coexpressed with Trx from an artificial operon; however, the TBD inserted retained a mesophilic binding affinity to Trx. The chimeric polymerase required 100 molar excess of Trx for processive polymerase activity at 60??C. TBD structural deformation at elevated temperatures was hypothesized to be the cause of the change in the subunit stoichiometry. Mutagenesis of TBD would be required to increase its thermostability. An efficient, rapid high throughput mutagenesis method (SLIM) was invented and would be appropriate for further studies.

Thesis (Ph.D.)--Tufts University, 2005. / Adviser: Krishna Kumar. Submitted to the Dept. of Chemistry. Includes bibliographical references. Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (D.Eng.)--University of Delaware, 2006. / Principal faculty advisor: Anne Skaja Robinson, Dept. of Chemical Engineering. Includes bibliographical references.

Undergraduate honors paper--Mount Holyoke College, 2008. Dept of Chemistry. / Includes bibliographical references (leaves 77-78).

Thesis (Ph. D.)--Ohio University, June, 2004. / Includes bibliographical references (leaves 128-139).

Thesis (PhD)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: David Pascual. Includes bibliographical references (leaves 278-316).