(276h) Highly Orthogonal Programmable Riboregulators for Complex Digital Logic in E. Coli | AIChE

(276h) Highly Orthogonal Programmable Riboregulators for Complex Digital Logic in E. Coli


Green, A. A. - Presenter, Harvard University
Collins, J. J., Howard Hughes Medical Institute, Boston University
Silver, P., Harvard Medical School
Yin, P., Wyss Institute

A diverse array of synthetic genetic programs has been constructed inside living cells to carry out digital logic operations1, record memories of biochemical events2, and synthesize valuable chemicals3,4. These behaviors have been established largely using existing biological components rewired in novel ways to enable new capabilities. It remains a significant challenge, however, to engineer living systems with functionalities that extend beyond rudimentary logic operations. This challenge arises in part from the limited number of biological circuit components that operate independently from one another and the difficulty in creating robust approaches to combine these elements for predictable behavior in living cells. Here, we exploit synthetic networks of RNA self-assembly reactions to construct a versatile platform for complex digital logic in Escherichia coli. The building blocks of this computational platform are newly designed programmable riboregulators of gene expression consisting of pairs of cis-repressed mRNAs (crRNAs) and trans-activating RNAs (taRNAs), which hybridize with their cognate crRNAs to de-repress them. In contrast to previous work, these RNA-based regulatory systems exploit design concepts derived not from existing biological systems5-7 but from hairpin-based strand displacement reactions developed in nucleic acid nanotechnology8,9. These design features greatly expand the RNA sequence space of potential riboregulators compared to earlier systems7,10 and provide the riboregulators with a high degree of programmability, orthogonality, and modularity for carrying out digital logic. To demonstrate these capabilities, we have assembled a family of 48 programmable riboregulators that increase expression of a GFP reporter by a factor of 40 or more upon transactivation and have validated a set of 15 highly orthogonal riboregulators with cross-talk levels below 10% in vivo. We have used this expansive toolkit of synthetic biological components to construct a number of complex decision-making systems in living cells. These include a multi-input OR gate that activates protein translation only in response to one of six programmed taRNAs and an AND circuit that produces GFP only upon assembly of a two-strand RNA complex. These programmable riboregulators provide a robust platform for evaluating more complex computations in living cells, and also constitute a promising test bed for probing interactions between assemblies of multiple RNA molecules in vivo.



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9.   Yin, P., Choi, H. M. T., Calvert, C. R. & Pierce, N. A., Nature 451, 318-322 (2008).

10.  Callura, J. M., Cantor, C. R. & Collins, J. J., Proc. Natl. Acad. Sci. USA 109, 5850-5855 (2012).