Chappell, James2024-01-242024-01-242023-122023-11-09December 2Liu, Baiyang. "RNA-based portable genetic circuits and synthetic plasmids." (2023). PhD diss., Rice University. https://hdl.handle.net/1911/115408https://hdl.handle.net/1911/115408EMBARGO NOTE: This item is embargoed until 2025-12-01The lack of modularity impedes the creation of synthetic biological systems at the same level of complexity as modern electronic systems, which can contain millions of logic gates. Recently, the development of novel RNA regulators poses an opportunity to overcome this challenge. In this thesis work, we aim to use RNA-based strategies to improve the modularity of biological systems on three different levels: chassis modularity, circuit modularity, and part modularity. Firstly, we verify the portability of Small Transcription Activating RNA (STAR) regulatory system in diverse Gram-negative bacteria. Then we break the chassis barrier by creating a regulatory RNA array design that allows for the construction of portable RNA-based circuits in multiple bacteria. Secondly, we propose an RNA compensation strategy to mitigate the retroactivity generated by the interconnection of multiple modules in RNA-based circuits. The insulation strategy is computationally proven to be effective not only in static modules but also in dynamic RNA-based circuits. Lastly, we develop synthetic plasmid origin of replication (SynOri) through the refactoring and reengineering of natural plasmid pMB1. The replication mechanism of pMB1 is replaced by pT181 RNA attenuator which allows the creation of modular plasmid library and plasmid replication-based circuits.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.RNAGenetic CircuitsSynthetic BiologyPlasmidThesis2024-01-24