Tracking, tuning, and terminating microbial physiology using synthetic riboregulators

Abstract

The development of biomolecular devices that interface with biological systems to reveal new insights and produce novel functions is one of the defining goals of synthetic biology. Our lab previously described a synthetic, riboregulator system that affords for modular, tunable, and tight control of gene expression in vivo. Here we highlight several experimental advantages unique to this RNA-based system, including physiologically relevant protein production, component modularity, leakage minimization, rapid response time, tunable gene expression, and independent regulation of multiple genes. We demonstrate this utility in four sets of in vivo experiments with various microbial systems. Specifically, we show that the synthetic riboregulator is well suited for GFP fusion protein tracking in wildtype cells, tight regulation of toxic protein expression, and sensitive perturbation of stress response networks. We also show that the system can be used for logic-based computing of multiple, orthogonal inputs, resulting in the development of a programmable kill switch for bacteria. This work establishes a broad, easy-to-use synthetic biology platform for microbiology experiments and biotechnology applications. S ynthetic biology seeks to reprogram organisms to alter natural biological behavior or perform novel functions, using engineered gene circuits, pathways, and whole genomes (1-8). Engineered gene circuits, in this regard, are typically constructed by assembling well-characterized biological components into unique architectures to achieve these unnatural capabilities, which are commonly used in biotechnology applications. A need also exists for synthetic biomolecular devices that interface with endogenous systems to track, probe, and influence, rather than reprogram, cellular physiology. Synthetic gene expression platforms that could accomplish this task would be well suited for use in a wide variety of phenotypic and genetic analyses aimed at expanding our knowledge of natural biomolecular components and networks, as well as enhancing our understanding of network dynamics under an array of conditions.In this work, we showcase an engineered riboregulator system that controls gene expression posttranscriptionally through highly specific RNA-RNA interactions (9). RNA molecules are useful components in synthetic biomolecular devices (10, 11) due to their large sequence space, the number of unique structures that can be adopted, the predictability of structure stability, and the range of functions enabled by these structures. Together, these features make RNA molecules ideal for interfacing with biological systems given that they can be used to control gene expression, sense biomolecules, and serve as foundational devices that can perform complex cellular behavior. As such, the engineered, RNA-based device space is remarkably diverse. Other successful examples include independent transcription-translation networks based on orthogonal mRNA-ribosome pairs (12), riboswitches in which RNA aptamers directly control translati...

Versatile RNA-sensing transcriptional regulators for engineering genetic networks

“…Recently, the diverse roles of RNA-mediated regulation have become important tools for synthetic biology applications ranging from detecting metabolic state (1), balancing metabolic pathway expression (2), tightly regulating toxin genes (3), and detecting environmentally harmful chemicals (4). In particular, RNA-based genetic parts have been engineered that regulate transcription through RNA-mediated transcription factor recruitment (5,6), transcript stability through small-molecule-mediated ribozyme cleavage (1,7) and siRNA targeted degradation (8), and translation through cis-acting mRNA conformational changes (9) and trans-acting antisense RNA-mRNA interactions (10,11).…”

Engineered Phagemids for Nonlytic, Targeted Antibacterial Therapies

“…This enables production of both targeted and broad-spectrum antibacterial treatments, depending upon bacteriophage selection. While some toxins tested here had little effect on the target E. coli strain, the selected toxins have a broad-range activity across many bacterial species [18][19][20][21][22]25 . Additionally, since our choices for antibacterial peptides are broad spectrum [11][12] , this system should provide a plug-and-play therapeutic that can be readily modified to suit its target and will therefore function in many target bacteria.…”

“…The first toxin, CcdB, is a topoisomerase inhibitor that interferes with DNA gyrase and results in the breakdown of bacterial DNA [18][19][20] , leading to cell death. YeeV is a toxin that inhibits cellular division by targeting two cytoskeletal proteins, FtsZ and MreB 21 ; however, this dual inhibition causes cells to 8 balloon and lyse, which is undesirable for our purposes.…”

Recent advances and versatility of MAGE towards industrial applications

“…In the past decade, synthetic biology has gained more scientific interest towards the building of synthetic circuits in a controllable and predictive manner (Purnick and Weiss 2009;Khalil and Collins 2010;Singh 2014). A number of synthetic devices and circuits such as riboregulators (Callura et al 2010;Klauser and Hartig 2013;Green et al 2014), riboswitches (Bayer and Smolke 2005;Wang et al 2008;Edwards et al 2010), oscillators (Elowitz and Leibler 2000;Stricker et al 2008;Tigges et al 2009;Mondragón-Palomino et al 2011) and biologic gates (Tamsir et al 2011;Ausländer et al 2012;Moon et al 2012) have been designed and characterized in a wide range of hosts.…”