Biological Circuits for Synthetic Biology
ScienceDaily (May 29, 2011) — "If you don't like the news, go out and make some of your own," said Wes "Scoop" Nisker. Taking a page from the book of San Francisco radio legend Scoop Nisker, biologists who find themselves dissatisfied with the microbes nature has provided are going out and making some of their own. Members of the fast-growing "synthetic biology" research community are designing and constructing novel organisms and biologically-inspired systems -- or redesigning existing organisms and systems -- to solve problems that natural systems cannot. The range of potential applications for synthetic biological systems runs broad and deep, and includes such profoundly important ventures as the microbial-based production of advanced biofuels and inexpensive versions of critical therapeutic drugs.
Berkeley Lab researchers are using RNA molecules to engineer genetic networks – analogous to microcircuits - into E. coli. (Credit: Image courtesy of DOE/Lawrence Berkeley National Laboratory)
Synthetic biology, however, is still a relatively new scientific field plagued with the trial and error inefficiencies that hamper most technologies in their early stages of development. To help address these problems, synthetic biologists aim to create biological circuits that can be used for the safer and more efficient construction of increasingly complex functions in microorganisms. A central component of such circuits is RNA, the multipurpose workhorse molecule of biology.
"A widespread natural ability to sense small molecules and regulate genes has made the RNA molecule an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering," says Adam Arkin, a computational biologist with the U.S. Department of Energy (DOE)'s Lawrence Berkeley National Laboratory (Berkeley Lab), where he serves as director of the Physical Biosciences Division. Arkin is also a professor at the University of California (UC) Berkeley where he directs the Synthetic Biology Institute, a partnership between UC Berkeley and Berkeley Lab.
In his multiple capacities, Arkin is leading a major effort to use RNA molecules for the engineering of programmable genetic networks. In recent years, scientists have learned that the behavior of cells is often governed by multiple different genes working together in networked teams that are regulated through RNA-based mechanisms. Synthetic biologists have been using RNA regulatory mechanisms to program genetic networks in cells to achieve specific results. However, to date these programming efforts have required proteins to propagate RNA regulatory signals. This can pose problems because one of the primary goals of synthetic biology is to create families of standard genetic parts that can be combined to create biological circuits with behaviors that are to some extent predictable. Proteins can be difficult to design and predict. They also add a layer of complexity to biological circuits that can delay and slow the dynamics of the circuit's responses.
"We're now able to eliminate the protein requirement and directly propagate regulatory signals as RNA molecules," Arkin says.
Working with their own variations of RNA transcription attenuators -- nucleotide sequences that under a specific set of conditions will stop the RNA transcription process -- Arkin and his colleagues engineered a system in which these independent attenuators can be configured to sense RNA input and synthesize RNA output signals. These variant RNA attenuators can also be configured to regulate multiple genes in the same cell and -- through the controlled expression of these genes -- perform logic operations.
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Versatile RNA-sensing transcriptional regulators for engineering genetic networks
Julius B. Lucksa, b,1,2, Lei Qia,1, Vivek K. Mutalik c, Denise Wang a, and Adam P. Arkin a,c,d,3
Author Affiliations
aDepartment of Bioengineering, University of California, Berkeley, CA 94720;
bMiller Institute for Basic Research in Science, Berkeley, CA 94720;
cPhysical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and
dCalifornia Institute for Quantitative Sciences (QB3), Berkeley, CA 94720
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.
gene networks, regulatory systems, orthogonal regulators
Footnotes
1J.B.L. and L.Q. contributed equally to this work.
2Present address: School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, NY 14850.
3To whom correspondence should be addressed at: E.O. Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS Stanley-922, Berkeley, CA 94720. E-mail: aparkin@lbl.gov.
Author contributions: J.B.L., L.Q., V.K.M., D.W., and A.P.A. designed research; J.B.L., L.Q., V.K.M., and D.W. performed research; J.B.L., L.Q., V.K.M., and A.P.A. analyzed data; and J.B.L., L.Q., V.K.M., and A.P.A. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
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This article has been corrected. See 
