Sequence-Level Design of Evolutionary-Robust Genetic Systems
Open Access
- Author:
- Reis, Alexander
- Graduate Program:
- Chemical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 12, 2020
- Committee Members:
- Howard M Salis, Dissertation Advisor/Co-Advisor
Howard M Salis, Committee Chair/Co-Chair
Phillip E Savage, Committee Member
Kristen Fichthorn, Committee Member
Philip C Bevilacqua, Outside Member
Phillip E Savage, Program Head/Chair - Keywords:
- synthetic biology
biophysics
CRISPR
genetic circuits
translation initiation - Abstract:
- This dissertation includes work towards the grand vision of complete sequence-level control in design of synthetic genetic systems, representing 3 high-impact, peer-reviewed publications from my time at Penn State University. Chapter 2 describes an automated model test system, SynBioMTS, for systematic development and improvement of sequence-function models (1). In this work, I use SynBioMTS to evaluate 6 existing bacterial translation initiation rate models, identify missing interactions that contribute to model error, and then develop RBS Calculator v2.1, the most accurate model to date. Chapter 3 describes an approach to solving the “DNA repeat challenge” (2). Repetitive DNA, although popular for its simplicity in design, can be problematic by causing DNA synthesis failure and genetic instability in vivo. I show that we can eliminate these issues by characterizing and using nonrepetitive versions of genetic parts such as E. coli σ70 promoters. Chapter 4 synergizes the efforts from Chapters 2 and 3 by designing what we call extra-long sgRNA arrays, or ELSAs, with CRISPRi for simultaneous, tunable regulation of dozens of genes in E. coli (3). We demonstrate ELSAs using three example applications including succinic acid overproduction, biocontainment by induced multiple auxotrophy, and induced antibiotic susceptibility. In all, this work enables more precise, tunable control for the design of evolutionary-robust synthetic genetic systems and lays the groundwork for further developments in sequence-level design.