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Genetic circuit design automation with Cello 2.0

Nature Protocols volume 17, pages 1097–1113 (2022)Cite this article

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Abstract

Cells interact with their environment, communicate among themselves, track time and make decisions through functions controlled by natural regulatory genetic circuits consisting of interacting biological components. Synthetic programmable circuits used in therapeutics and other applications can be automatically designed by computer-aided tools. The Cello software designs the DNA sequences for programmable circuits based on a high-level software description and a library of characterized DNA parts representing Boolean logic gates. This process allows for design specification reuse, modular DNA part library curation and formalized circuit transformations based on experimental data. This protocol describes Cello 2.0, a freely available cross-platform software written in Java. Cello 2.0 enables flexible descriptions of the logic gates’ structure and their mathematical models representing dynamic behavior, new formal rules for describing the placement of gates in a genome, a new graphical user interface, support for Verilog 2005 syntax and a connection to the SynBioHub parts repository software environment. Collectively, these features expand Cello’s capabilities beyond Escherichia coli plasmids to new organisms and broader genetic contexts, including the genome. Designing circuits with Cello 2.0 produces an abstract Boolean network from a Verilog file, assigns biological parts to each node in the Boolean network, constructs a DNA sequence and generates highly structured and annotated sequence representations suitable for downstream processing and fabrication, respectively. The result is a sequence implementing the specified Boolean function in the organism and predictions of circuit performance. Depending on the size of the design space and users’ expertise, jobs may take minutes or hours to complete.

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Fig. 1: Basic software architecture of Cello 2.0.
Fig. 2: NOR gate architectures.
Fig. 3: Visualization of gate model and function definitions.
Fig. 4: A selection of Eugene rules and circuits that satisfy or violate the rules.
Fig. 5: The Verilog editor and input and output selection panes in the GUI.
Fig. 6: The results view in the GUI.
Fig. 7: An XNOR gate designed in all five libraries available in Cello 2.0.
Fig. 8: The expected results of a case study aiming at designing the circuit 0×01 (three-input AND gate) using the SC1C1G1T1 library.

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Data availability

The data that support the findings of this study (i.e., standard UCF libraries, input sensor files and output device files) are openly available at https://doi.org/10.5281/zenodo.4676314.

Code availability

Code for Cello 2.0 is divided among various openly available repositories. The core circuit design module is available at https://doi.org/10.5281/zenodo.4676314, the code for the web application is available at https://doi.org/10.5281/zenodo.4676310 and the source code of the GUI (which is compiled into the webapp) is available at https://doi.org/10.5281/zenodo.4676300. A file (in .zip format), containing all the source code in the version used in the study, associated test data, parameters and documentation, is openly available at https://publication-artifacts.s3.amazonaws.com/cellov2.zip. The source code is openly distributed in accordance with Boston University’s Data Protection Standards (https://www.bu.edu/policies/data-protection-standards/pdf/) and under the MIT license at https://opensource.org/licenses/MIT.

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Acknowledgements

This work was supported by funding from DARPA award FA8750-17-C-0229 ‘Synergistic Discovery and Design (SD2)’ (D.D., C.A.V., C.J.M., T.S.J. and S.M.D.O.), DARPA award HR011-12-C-0067 ‘Living Foundries: 1000 Molecules’ (C.A.V), US Department of Energy award DE-SC0018368 (C.A.V.), NSF Synthetic Biology Engineering Research Center SA5284-11210 (C.A.V.) and NSF Grant No. 1522074 ‘The Living Computing Project’ (D.D., C.J.M., T.S.J. and S.M.D.O.). The authors also acknowledge the support of William Jackson from D.D.’s CIDAR Lab in providing programming assistance and reviewing the manuscript.

Author information

Authors and Affiliations

  1. Biological Design Center, Boston University, Boston, MA, USA

    Timothy S. Jones, Samuel M. D. Oliveira & Douglas Densmore

  2. Department of Electrical and Computer Engineering, Boston University, Boston, MA, USA

    Timothy S. Jones, Samuel M. D. Oliveira & Douglas Densmore

  3. Electrical, Computer & Energy Engineering, University of Colorado Boulder, Boulder, CO, USA

    Chris J. Myers

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

    Christopher A. Voigt

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Contributions

C.A.V. and D.D. conceived and supervised the project. C.J.M. provided supervision and technical assistance with SBOL and SynBioHub integrations. S.M.D.O assisted with input files and UCF designs and preparation. T.S.J. wrote the Cello 2.0 software. All authors read and revised the manuscript.

Corresponding author

Correspondence to Douglas Densmore.

Ethics declarations

Competing interests

D.D. and C.A.V. are co-founders of Asimov. Asimov is a company that uses software to engineer biology.

Peer review

Peer review information

Nature Protocols thanks Mark Isalan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Nielsen, A. et al. Science 352, aac7341 (2016): https://doi.org/10.1126/science.aac7341

Chen, Y. et al. Nat. Microbiol. 5, 1349–1360 (2020): https://doi.org/10.1038/s41564-020-0757-2

Vaidyanathan, P. et al. Proc. IEEE 11, 2196–2207 (2015): https://doi.org/10.1109/JPROC.2015.2443832

Key data used in this protocol

Taketani, M. et al. Nat. Biotechnol. 38, 962–969 (2020): https://doi.org/10.1038/s41587-020-0468-5

Supplementary information

Supplementary Information

Supplementary Manual, Supplementary Listings 1–35 and Supplementary Figs. 1–6.

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Jones, T.S., Oliveira, S.M.D., Myers, C.J. et al. Genetic circuit design automation with Cello 2.0. Nat Protoc 17, 1097–1113 (2022). https://doi.org/10.1038/s41596-021-00675-2

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