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Programming Bacterial Gene Expression Using Synthetic CRISPR-Cas Transcriptional Regulators
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| 자료유형 | 학위논문 |
|---|---|
| 서명/저자사항 | Programming Bacterial Gene Expression Using Synthetic CRISPR-Cas Transcriptional Regulators. |
| 개인저자 | Dong, Chen. |
| 단체저자명 | University of Washington. Chemistry. |
| 발행사항 | [S.l.]: University of Washington., 2019. |
| 발행사항 | Ann Arbor: ProQuest Dissertations & Theses, 2019. |
| 형태사항 | 221 p. |
| 기본자료 저록 | Dissertations Abstracts International 81-04B. Dissertation Abstract International |
| ISBN | 9781088330395 |
| 학위논문주기 | Thesis (Ph.D.)--University of Washington, 2019. |
| 일반주기 |
Source: Dissertations Abstracts International, Volume: 81-04, Section: B.
Advisor: Zalatan, Jesse George. |
| 이용제한사항 | This item must not be sold to any third party vendors.This item must not be added to any third party search indexes. |
| 요약 | Bacteria play a central role in biosynthesis to produce value-added organic chemicals due to its diverse carbon and energy source preferences. Implementing synthetic transcriptional regulation devices can advance our ability to modify gene expression in bacteria for engineering production strains. The CRISPR-Cas activation (CRISPRa) system, a programmable transcriptional activator with wide applications in eukaryotic organisms, has been under-utilized in bacterial due to the lack of efficient transcriptional activation domains. This work describes our contribution to the development and understanding of bacterial CRISPR-Cas-based transcriptional regulation devices. We screened novel bacterial activation domains to be used as CRISPR-Cas activators in E. coli, and optimized our strongest activation domain, SoxS into a programmable CRISPR activator. In addition, we investigated the properties of the well-established CRISPRi repression system and found that partial repression can be achieved by controlling the expression level of the sgRNA. Combining CRISPRa and CRISPRi, we demonstrated inducible simultaneous up- and down-regulation of a dual reporter gene and the CRISPR-mediated regulation of the ethanol bioproduction pathway. Moreover, we also learned important properties of the bacterial CRISPRa system which is much more restrictive than the eukaryotic CRISPRa system. CRISPRa activity is dependent on the sigma factor that the promoter recruits, the baseline strength of the promoter, the sequence composition of the promoter, the presence of nearby transcription factors, and the precise positioning of the scRNA target. We attempted to relieve these restrictions by implementing an engineered Cas protein that can bind to a wider range of targets. Lastly, we describe our efforts to transport the CRISPRa system into other non-E.coli bacteria. CRISPR-SoxS activator proved to be active in Pseudomonas putida, but not in the other organisms we tested. Therefore, we propose to characterize host-specific activation domains for bacteria whose cellular machineries are incompatible with SoxS. Together, this work provided a novel programmable gene activation device in bacteria and sets up the foundation for the development of complex, broad-host-range bacterial cellular devices for biosynthetic applications. |
| 일반주제명 | Molecular biology. Bioengineering. Gene expression. Biochemistry. Biosynthesis. Bacteria. CRISPR. |
| 언어 | 영어 |
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: 이 자료의 원문은 한국교육학술정보원에서 제공합니다. |
