Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 143-159). / The ability to engineer complex genetic programs could have a huge impact on many industries, yielding organisms that can respond to their environment and perform functions relevant to manufacturing, agriculture, and medicine. However, such engineering efforts have proven difficult, in part because these programs often require precise levels of gene expression for proper function. It is especially tough to build programs that have robust activity, as any changes to the host cells can perturb the context of the genetic system and disrupt carefully tuned expression levels. Additionally, genetic programs often place high demands on host resources, which can adversely affect cell growth and further upset the intended function. In this thesis, we describe two regulatory systems in Escherichia coli that could serve to separate synthetic genetic programs from their host context, potentially leading to more robust activity. First, we build a 'resource allocator' by fragmenting T7 RNA polymerase variants into a conserved fragment and a set of variable fragments. The resource allocator limits the total number of polymerases that can be active in a genetic program, with the aim of protecting the host from being overburdened. This transcriptional budget can be allocated to different elements of the genetic program as necessary and further regulated using additional protein fragments. Second, we demonstrate a set of stabilized promoters that can maintain a level of gene expression independent of their genetic context. These promoters utilize a noncooperative incoherent feedforward loop to buffer differences in gene expression caused by changes in copy number. We demonstrate that stabilized promoters can be moved between plasmids and different locations on the genome with little change in expression. Further, they minimize the effects of other perturbations that can affect copy number, such as genome mutations and media composition. / by Thomas Hale Segall-Shapiro. / Ph. D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/113965 |
| Date | January 2017 |
| Creators | Segall-Shapiro, Thomas Hale |
| Contributors | Christopher A. Voigt., Massachusetts Institute of Technology. Department of Biological Engineering., Massachusetts Institute of Technology. Department of Biological Engineering. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 159 pages, application/pdf |
| Rights | MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission., http://dspace.mit.edu/handle/1721.1/7582 |
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