Versatile RNA-sensing transcriptional regulators for engineering genetic networks
Edited* by Jennifer A. Doudna, University of California, Berkeley, CA, and approved April 11, 2011 (received for review October 19, 2010)
Abstract
The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.
Acknowledgments.
The authors thank Richard Novick (Department of Microbiology, New York University School of Medicine, New York) for donating plasmid pT181; Ron Breaker, Blake Wiedenheft, and Vincent Rouilly for comments; Weston Whitaker and Jeff Skerker for discussions; and Richard Shan and Quintara Biosciences for assistance in plasmid sequencing. Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract DE-AC02-05CH11231. This work was supported by the Synthetic Biology Engineering Research Center under National Science Foundation Grant 04-570/0540879. J.B.L. acknowledges the financial support of the Miller Institute for Basic Research in Science.
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Published online: May 9, 2011
Published in issue: May 24, 2011
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Acknowledgments
The authors thank Richard Novick (Department of Microbiology, New York University School of Medicine, New York) for donating plasmid pT181; Ron Breaker, Blake Wiedenheft, and Vincent Rouilly for comments; Weston Whitaker and Jeff Skerker for discussions; and Richard Shan and Quintara Biosciences for assistance in plasmid sequencing. Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract DE-AC02-05CH11231. This work was supported by the Synthetic Biology Engineering Research Center under National Science Foundation Grant 04-570/0540879. J.B.L. acknowledges the financial support of the Miller Institute for Basic Research in Science.
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*This Direct Submission article had a prearranged editor.
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The authors declare no conflict of interest.
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Versatile RNA-sensing transcriptional regulators for engineering genetic networks, Proc. Natl. Acad. Sci. U.S.A.
108 (21) 8617-8622,
https://doi.org/10.1073/pnas.1015741108
(2011).
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