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Automated design of thousands of nonrepetitive parts for engineering stable genetic systems

Nature Biotechnology volume 38, pages 1466–1475 (2020)Cite this article

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Abstract

Engineered genetic systems are prone to failure when their genetic parts contain repetitive sequences. Designing many nonrepetitive genetic parts with desired functionalities remains a difficult challenge with high computational complexity. To overcome this challenge, we developed the Nonrepetitive Parts Calculator to rapidly generate thousands of highly nonrepetitive genetic parts from specified design constraints, including promoters, ribosome-binding sites and terminators. As a demonstration, we designed and experimentally characterized 4,350 nonrepetitive bacterial promoters with transcription rates that varied across a 820,000-fold range, and 1,722 highly nonrepetitive yeast promoters with transcription rates that varied across a 25,000-fold range. We applied machine learning to explain how specific interactions controlled the promoters’ transcription rates. We also show that using nonrepetitive genetic parts substantially reduces homologous recombination, resulting in greater genetic stability.

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Fig. 1: Nonrepetitive genetic parts and their activities.
Fig. 2: The Nonrepetitive Parts Calculator.
Fig. 3: Design and characterization of 4,350 nonrepetitive bacterial promoters.
Fig. 4: Design and characterization of 1,722 nonrepetitive yeast promoters.
Fig. 5: Analysis of nonrepetitive bacterial promoters.
Fig. 6: Sequence determinants of nonrepetitive yeast promoters.

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Data availability

All characterized genetic part sequences and measurements are provided in the Supplementary Information.

Code availability

A user-friendly interface to the Nonrepetitive Parts Calculator is available at https://salislab.net/software. Source code is available at https://github.com/hsalis/SalisLabCode.

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Acknowledgements

This project was supported by funds from the Air Force Office of Scientific Research (grant no. FA9550-14-1-0089), the Defense Advanced Research Projects Agency (grant nos. FA8750-17-C-0254 and HR001117C0095), the Department of Energy (grant no. DE-SC0019090), and a Graduate Research Innovation award to A.H. from the Huck Institutes of the Life Sciences.

Author information

Authors and Affiliations

  1. Bioinformatics and Genomics, Pennsylvania State University, University Park, PA, USA

    Ayaan Hossain & Howard M. Salis

  2. Department of Electrical & Computer Engineering, University of Washington, Seattle, WA, USA

    Eriberto Lopez, Devin Strickland & Eric Klavins

  3. Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA

    Sean M. Halper, Daniel P. Cetnar, Alexander C. Reis & Howard M. Salis

  4. Department of Biological Engineering, Pennsylvania State University, University Park, PA, USA

    Howard M. Salis

  5. Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, USA

    Howard M. Salis

Authors
  1. Ayaan Hossain
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  2. Eriberto Lopez
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Contributions

A.H. and H.M.S. conceived the study. A.H., E.L., D.P.C., S.M.H., A.C.R. and D.S. designed and carried out the experiments. A.H., A.C.R. and H.M.S. developed the algorithms and performed the data analysis. A.H., D.S., E.K. and H.M.S. wrote the manuscript.

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Correspondence to Howard M. Salis.

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Supplementary information

Supplementary Information

Reporting Summary

Supplementary Data 1

Existing genetic parts and their repetitiveness.

Supplementary Data 2

Toolboxes of nonrepetitive genetic parts.

Supplementary Data 3

Sequences and measurements for nonrepetitive bacterial and yeast promoters.

Supplementary Data 4

Model features for the nonrepetitive yeast promoters.

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Hossain, A., Lopez, E., Halper, S.M. et al. Automated design of thousands of nonrepetitive parts for engineering stable genetic systems. Nat Biotechnol 38, 1466–1475 (2020). https://doi.org/10.1038/s41587-020-0584-2

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