Design and Construction of Generalizable RNA-Protein Hybrid Controllers by Level-Matched Genetic Signal Amplification

Abstract

Summary For synthetic biology applications, protein-based transcriptional genetic controllers are limited in terms of orthogonality, modularity, and portability. Although ribozyme-based switches can address these issues, their current two-stage architectures and limited dynamic range hinder their broader incorporation into systems-level genetic controllers. Here, we address these challenges by implementing an RNA-protein hybrid controller with a three-stage architecture that introduces a transcription-based amplifier between an RNA sensor and a protein actuator. To facilitate the construction of these more complex circuits, we use a model-guided strategy to efficiently match the activities of stages. The presence of the amplifier enabled the three-stage controller to have up to 200-fold higher gene expression than its two-stage counterpart and made it possible to implement higher-order controllers, such as multilayer Boolean logic and feedback systems. The modularity inherent in the three-stage architecture along with the sensing flexibility of RNA devices presents a generalizable framework for designing and building sophisticated genetic control systems.

Computational design of small transcription activating RNAs for versatile and dynamic gene regulation

“…In addition we showed that combining STARs with inducible promoter systems could tune their transfer functions, which to date has been largely achieved through engineering of DNA promoters and protein transcription factors 53 . Thus STARs add to an increasingly powerful toolbox of regulators that can be used in composite with existing regulatory mechanisms to alter their regulatory properties 54 , 55 .…”

Engineering a Functional small RNA Negative Autoregulation Network with Model-guided Design

“…Specifically, synthetic biologists have engineered a diverse array of RNA regulators that can be used to control many of the core processes of gene expression including transcription [2][3][4][5] , translation [6][7][8] and mRNA degradation [9][10][11][12] . In addition to being able to tune the expression of individual genes, these engineered RNA regulators have also been connected together to create synthetic gene networks in the form of logic gates 2,3,5,7,13 , cascades 2,5,[13][14][15][16][17] , single input modules 5,16 feed forward loops 18 , and hybrid feedback controllers 12 . While these synthetic network motifs showcase some of the advantages of RNA regulators, they represent the beginning of larger efforts to create a greater diversity of RNA network function that is necessary to engineer higher order cellular behavior.…”

A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

“…RNA biosensors have been used as genetically-encoded controllers for a variety of synthetic biology applications 41,42 . Expanding the diversity of ligand-responsive RNA switches can increase the number and complexity of logical operations in biological circuits [43][44][45] . In biomanufacturing, biosensors that can monitor the accumulation of intermediate or product metabolites can be applied to generate screens of selections for improved enzyme activity or pathway flux 15,46 or to implement dynamic feedback control 45 .…”

“…Expanding the diversity of ligand-responsive RNA switches can increase the number and complexity of logical operations in biological circuits [43][44][45] . In biomanufacturing, biosensors that can monitor the accumulation of intermediate or product metabolites can be applied to generate screens of selections for improved enzyme activity or pathway flux 15,46 or to implement dynamic feedback control 45 . Biosensors that responds to small molecule drugs with safe pharmacological profiles (e.g., aciclovir, gardiquimod) can enable the generation of conditional genetic controllers for CRISPR/Cas9-based therapies 29 , gene therapies 47 , and cell therapies 48,49 , to mitigate toxic effects of prolonged transgene expression or cell activation 12 .…”