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Molecular Bioengineering: From Protein Stability to Population Suicide

dc.contributor.advisor Hellinga, Homme W
dc.contributor.advisor You, Lingchong
dc.contributor.author Marguet, Philippe Robert
dc.date.accessioned 2011-01-06T16:04:00Z
dc.date.available 2011-01-06T16:04:00Z
dc.date.issued 2010
dc.identifier.uri https://hdl.handle.net/10161/3143
dc.description.abstract <p>Driven by the development of new technologies and an ever expanding knowledge base of molecular and cellular function, Biology is rapidly gaining the potential to develop into a veritable engineering discipline - the so-called `era of synthetic biology' is upon us. Designing biological systems is advantageous because the engineer can leverage existing capacity for self-replication, elaborate chemistry, and dynamic information processing. On the other hand these functions are complex, highly intertwined, and in most cases, remain incompletely understood. Brazenly designing within these systems, despite large gaps in understanding, engenders understanding because the design process itself highlights gaps and discredits false assumptions. </p><p>Here we cover results from design projects that span several scales of complexity. First we describe the adaptation and experimental validation of protein functional assays on minute amounts of material. This work enables the application of cell-free protein expression tools in a high-throughput protein engineering pipeline, dramatically increasing turnaround time and reducing costs. The parts production pipeline can provide new building blocks for synthetic biology efforts with unprecedented speed. Tools to streamline the transition from the in vitro pipeline to conventional cloning were also developed. Next we detail an effort to expand the scope of a cysteine reactivity assay for generating information-rich datasets on protein stability and unfolding kinetics. We go on to demonstrate how the degree of site-specific local unfolding can also be determined by this method. This knowledge will be critical to understanding how proteins behave in the cellular context, particularly with regards to covalent modification reactions. Finally, we present results from an effort to engineer bacterial cell suicide in a population-dependent manner, and show how an underappreciated facet of plasmid physiology can produce complex oscillatory dynamics. This work is a prime example of engineering towards understanding.</p>
dc.subject Biology, Molecular
dc.subject Chemistry, Biochemistry
dc.subject Engineering, Biomedical
dc.subject gene circuits
dc.subject local unfolding
dc.subject protein engineering
dc.subject protein stability
dc.subject quantitative cysteine reactivity
dc.subject synthetic biology
dc.title Molecular Bioengineering: From Protein Stability to Population Suicide
dc.type Dissertation
dc.department Biochemistry


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