Abstract
Bacterial regulators of transcriptional elongation are versatile units for building custom genetic switches, as they control the expression of both coding and noncoding RNAs, act on multigene operons and can be predictably tethered into higher-order regulatory functions (a property called composability). Yet the less versatile bacterial regulators of translational initiation are substantially easier to engineer. To bypass this tradeoff, we have developed an adaptor that converts regulators of translational initiation into regulators of transcriptional elongation in Escherichia coli. We applied this adaptor to the construction of several transcriptional attenuators and activators, including a small molecule–triggered attenuator and a group of five mutually orthogonal riboregulators that we assembled into NOR gates of two, three or four RNA inputs. Continued application of our adaptor should produce large collections of transcriptional regulators whose inherent composability can facilitate the predictable engineering of complex synthetic circuits.
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Acknowledgements
We thank C. Yanofsky and M. Samoilov for thoughtful discussions, D. Chen for experimental assistance and S. Meyer for assistance with SHAPE experiments. This work was funded by the US National Science Foundation as part of the Synthetic Biology Engineering Research Center (A.P.A.), the Miller Institute for Basic Scientific Research (C.C.L. and J.B.L.) and the US Department of Defense through the National Defense Science and Engineering Graduate Fellowship (T.H.S.-S.).
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C.C.L. conceived of the study, and A.P.A. advised in all aspects of the study. All authors were involved in designing the experiments. C.C.L., L.Q., J.B.L., T.H.S.-S. and V.K.M. performed experiments and interpreted the data. L.Q. and D.W. conducted the NOR gate experiments and the aptamer fusion experiments. J.B.L. conducted and analyzed the SHAPE experiments. C.C.L., L.Q. and A.P.A. wrote the manuscript. All authors discussed results and commented on the manuscript.
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US Provisional Patent number 61/540,413 is pending on this work.
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Liu, C., Qi, L., Lucks, J. et al. An adaptor from translational to transcriptional control enables predictable assembly of complex regulation. Nat Methods 9, 1088–1094 (2012). https://doi.org/10.1038/nmeth.2184
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DOI: https://doi.org/10.1038/nmeth.2184
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