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Designable DNA-binding domains enable construction of logic circuits in mammalian cells

Abstract

Electronic computer circuits consisting of a large number of connected logic gates of the same type, such as NOR, can be easily fabricated and can implement any logic function. In contrast, designed genetic circuits must employ orthogonal information mediators owing to free diffusion within the cell. Combinatorial diversity and orthogonality can be provided by designable DNA- binding domains. Here, we employed the transcription activator–like repressors to optimize the construction of orthogonal functionally complete NOR gates to construct logic circuits. We used transient transfection to implement all 16 two-input logic functions from combinations of the same type of NOR gates within mammalian cells. Additionally, we present a genetic logic circuit where one input is used to select between an AND and OR function to process the data input using the same circuit. This demonstrates the potential of designable modular transcription factors for the construction of complex biological information-processing devices.

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Figure 1: Design of single-layer genetic NOR gates based on orthogonal designed TALE repressors in mammalian cells.
Figure 2: Construction of the XOR logic gate from NOR gates based on the orthogonal designed TALE repressors.
Figure 3: Implementation of all 16 two-input Boolean logic functions constructed from combinations of designed TALE repressor-based NOR gates.
Figure 4: Design of the genetic circuit that allows selection of logic function processing data inputs.

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Acknowledgements

This research study was supported by the program and projects from the Slovenian Research Agency (P4-0176 and N5-0003 to R.J.) and the EN-FIST Centre of Excellence financed in part by the European structural funds. We acknowledge the members and mentors of the 2012 Slovenian International Genetically Engineered Machine (iGEM) Team (U. Bezeljak, V. Forstnerič, A. Golob, M. Jerala, L. Kadunc, J. Lonzarić, Z. Lužnik, A. Oblak, F. Pavlovec, B. Pirč, A. Smole, M. Somrak, M. Stražar, D. Vučko and U. Zupančič) for their inspiration and help in the development of TALE-based regulation. We thank M. Fussenegger (Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Zurich) for plasmids for erythromycin- and pristinamycin-inducible systems.

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Contributions

R.G., T.L., M.B. and A.M. performed and analyzed the experiments; B.Š., A.D. and R.G. designed the logic gates and analyzed the triple gate circuits; R.J. designed the study and wrote the manuscript; and M.B., R.G., B.Š. and A.D. helped in writing the manuscript.

Corresponding author

Correspondence to Roman Jerala.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1–14, Supplementary Note and Supplementary Tables 1–11. (PDF 2980 kb)

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Gaber, R., Lebar, T., Majerle, A. et al. Designable DNA-binding domains enable construction of logic circuits in mammalian cells. Nat Chem Biol 10, 203–208 (2014). https://doi.org/10.1038/nchembio.1433

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