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Genomic mining of prokaryotic repressors for orthogonal logic gates

Nature Chemical Biology volume 10, pages 99–105 (2014)Cite this article

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Abstract

Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply 'part mining' to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method, and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and exhibit minimal interactions with other promoters. Each repressor-promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOT/NOR gates, there are >1054 circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.

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Figure 1: A large repressor library is compiled using genome mining.
Figure 2: The identification of operator sequences using an in vitro array assay.
Figure 3: Design and screening of orthogonal promoters.
Figure 4: Response function measurement.
Figure 5: Construction and characterization of integrated circuits.

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Acknowledgements

We would like to thank C. Warren of Illumavista Biosciences for aid with array design, array experiments, data analysis and Perl scripts that were used for array data extraction. Research for this publication was conducted with government support under FA9550-11-C-0028 and was awarded by the Department of Defense, Air Force Office of Scientific Research, National Defense Science and Engineering Graduate Fellowship, 32 CFR 168a to A.A.K.N. C.A.V. is supported by Life Technologies, Defense Advanced Research Projects Agency Chronicle of Lineage Indicative of Origins (DARPA CLIO) (N66001-12-C-4016), Office of Naval Research (N00014-13-1-0074), US National Institutes of Health (GM095765) and the US National Science Foundation Synthetic Biology Engineering Research Center (SynBERC, SA5284-11210).

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Authors and Affiliations

  1. Department of Biological Engineering, Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Brynne C Stanton, Alec A K Nielsen & Christopher A Voigt

  2. Department of Biochemistry and Biophysics, University of California–San Francisco, San Francisco, California, USA

    Alvin Tamsir

  3. Synthetic Biology R&D Unit, Life Technologies, Carlsbad, California, USA

    Kevin Clancy & Todd Peterson

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  1. Brynne C Stanton
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Contributions

C.A.V., B.C.S. and A.A.K.N. designed and performed experiments, analyzed data and wrote the manuscript. A.T. designed experiments. K.C. aided in project management. T.P. aided in project management.

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Correspondence to Christopher A Voigt.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–16 and Supplementary Tables 1–7. (PDF 2150 kb)

Supplementary Data Set 1

Supplementary Repressor Library Sequence Table (XLSX 64 kb)

Supplementary Data Set 2

Supplementary Repressor Motif Array Data (XLSX 62 kb)

Supplementary Data Set 3

Supplementary Promoter Library Cytometry Data (XLSX 2719 kb)

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Stanton, B., Nielsen, A., Tamsir, A. et al. Genomic mining of prokaryotic repressors for orthogonal logic gates. Nat Chem Biol 10, 99–105 (2014). https://doi.org/10.1038/nchembio.1411

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