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Genetic programs constructed from layered logic gates in single cells

Abstract

Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics1. These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits2,3,4,5,6, and they have been used in various cellular engineering applications, including the implementation of process control in metabolic networks and the coordination of spatial differentiation in artificial tissues. A key limitation is that the circuits are based on biochemical interactions occurring in the confined volume of the cell, so the size of programs has been limited to a few circuits1,7. Here we apply part mining and directed evolution to build a set of transcriptional AND gates in Escherichia coli. Each AND gate integrates two promoter inputs and controls one promoter output. This allows the gates to be layered by having the output promoter of an upstream circuit serve as the input promoter for a downstream circuit. Each gate consists of a transcription factor that requires a second chaperone protein to activate the output promoter. Multiple activator–chaperone pairs are identified from type III secretion pathways in different strains of bacteria. Directed evolution is applied to increase the dynamic range and orthogonality of the circuits. These gates are connected in different permutations to form programs, the largest of which is a 4-input AND gate that consists of 3 circuits that integrate 4 inducible systems, thus requiring 11 regulatory proteins. Measuring the performance of individual gates is sufficient to capture the behaviour of the complete program. Errors in the output due to delays (faults), a common problem for layered circuits, are not observed. This work demonstrates the successful layering of orthogonal logic gates, a design strategy that could enable the construction of large, integrated circuits in single cells.

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Figure 1: Mining circuits from genomic islands.
Figure 2: Part engineering to improve dynamic range and orthogonality.
Figure 3: Three 2-input AND gates constructed using Salmonella (left), Shigella (middle) and Pseudomonas (right) parts.
Figure 4: Genetic programs formed by layering AND gates.
Figure 5: Performance of genetic programs.

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Acknowledgements

C.A.V. is supported by Life Technologies, Defense Advanced Research Projects Agency Chronicle of Lineage Indicative of Origins (DARPA; CLIO N66001-12-C-4018), the Office of Naval Research (N00014-10-1-0245), the National Science Foundation (NSF; CCF-0943385), the National Institutes of Health (AI067699) and the NSF Synthetic Biology Engineering Research Center (SynBERC; SA5284-11210). The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred.

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T.S.M. designed and performed the experiments, analysed the data, developed the computational models and wrote the manuscript. C.L. developed the computational models. A.T. analysed the data. B.C.S. performed experiments. C.A.V. designed experiments, analysed the data, developed the computational models and wrote the manuscript.

Corresponding author

Correspondence to Christopher A. Voigt.

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The authors declare no competing financial interests.

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This file contains Supplementary Text and Data, Supplementary Tables 1-5, Supplementary Figures 1-18 and Supplementary References – see Supplementary Contents page for further details. (PDF 961 kb)

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Moon, T., Lou, C., Tamsir, A. et al. Genetic programs constructed from layered logic gates in single cells. Nature 491, 249–253 (2012). https://doi.org/10.1038/nature11516

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