Characterization of 582 natural and synthetic terminators and quantification of their design constraints


Large genetic engineering projects require more cistrons and consequently more strong and reliable transcriptional terminators. We have measured the strengths of a library of terminators, including 227 that are annotated in Escherichia coli—90 of which we also tested in the reverse orientation—and 265 synthetic terminators. Within this library we found 39 strong terminators, yielding >50-fold reduction in downstream expression, that have sufficient sequence diversity to reduce homologous recombination when used together in a design. We used these data to determine how the terminator sequence contributes to its strength. The dominant parameters were incorporated into a biophysical model that considers the role of the hairpin in the displacement of the U-tract from the DNA. The availability of many terminators of varying strength, as well as an understanding of the sequence dependence of their properties, will extend their usability in the forward design of synthetic cistrons.

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Figure 1: Measurement of terminator strength for the natural and synthetic libraries.
Figure 2: Sequence features that contribute to terminator strength.
Figure 3: Impact of changing the various components of a terminator in three strong terminator 'scaffolds'.
Figure 4: Biophysical model of terminator strength, TS, and recombination propensity.


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C.A.V., P.L., A.A.K.N., J.A.N.B. and Y.-J.C. are supported by Life Technologies and the US National Science Foundation Synthetic Biology Engineering Research Center (SynBERC). Y.-J.C. thanks the PhRMA Foundation for a Postdoctoral Fellowship in Informatics. We thank R. Landick and D.Y. Zhang for their advanced review for the manuscript.

Author information




C.A.V. conceived and supervised the project. Y.-J.C., A.A.K.N. and J.A.N.B. designed and performed the experiments. C.A.V., P.L. and Y.-J.C. constructed the biophysical model. K.C. and T.P. oversaw the project. C.A.V., Y.-J.C. and P.L. wrote the manuscript.

Corresponding author

Correspondence to Christopher A Voigt.

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Competing interests

K.C. and T.P. are employees of Life Technologies, which funded this work and commercializes the terminator libraries and assays described.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–23, Supplementary Table 1 and Supplementary Notes 1–11 (PDF 1755 kb)

Supplementary Table 2

Natural terminators (XLSX 130 kb)

Supplementary Table 3

Synthetic terminators (XLSX 94 kb)

Supplementary Table 4

Removed terminators (XLSX 18 kb)

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Chen, YJ., Liu, P., Nielsen, A. et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods 10, 659–664 (2013).

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