Browsing by Author "Liu, Baiyang"

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    A portable regulatory RNA array design enables tunable and complex regulation across diverse bacteria
    (Springer Nature, 2023) Liu, Baiyang; Samaniego, Christian Cuba; Bennett, Matthew R.; Franco, Elisa; Chappell, James; Bioengineering; Biosciences
    A lack of composable and tunable gene regulators has hindered efforts to engineer non-model bacteria and consortia. Toward addressing this, we explore the broad-host potential of small transcription activating RNA (STAR) and propose a design strategy to achieve tunable gene control. First, we demonstrate that STARs optimized for E. coli function across different Gram-negative species and can actuate using phage RNA polymerase, suggesting that RNA systems acting at the level of transcription are portable. Second, we explore an RNA design strategy that uses arrays of tandem and transcriptionally fused RNA regulators to precisely alter regulator concentration from 1 to 8 copies. This provides a simple means to predictably tune output gain across species and does not require access to large regulatory part libraries. Finally, we show RNA arrays can be used to achieve tunable cascading and multiplexing circuits across species, analogous to the motifs used in artificial neural networks.
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    RNA-based portable genetic circuits and synthetic plasmids
    (2023-11-09) Liu, Baiyang; Chappell, James
    The 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.