Genetic circuit design automation with Cello 2.0.

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.

Genetic circuit design automation for yeast.

Cells can be programmed to monitor and react to their environment using genetic circuits. Design automation software maps a desired circuit function to a DNA sequence, a process that requires units of gene regulation (gates) that are simple to connect and behave predictably. This poses a challenge for eukaryotes due to their complex mechanisms of transcription and translation. To this end, we have developed gates for yeast (Saccharomyces cerevisiae) that are connected using RNA polymerase flux as the signal carrier and are insulated from each other and host regulation. They are based on minimal constitutive promoters (~120 base pairs), for which rules are developed to insert operators for DNA-binding proteins. Using this approach, we constructed nine NOT/NOR gates with nearly identical response functions and 400-fold dynamic range. In circuits, they are transcriptionally insulated from each other by placing ribozymes downstream of terminators to block nuclear export of messenger RNAs resulting from RNA polymerase readthrough. Based on these gates, Cello 2.0 was used to build circuits with up to 11 regulatory proteins. A simple dynamic model predicts the circuit response over days. Genetic circuit design automation for eukaryotes simplifies the construction of regulatory networks as part of cellular engineering projects, whether it be to stage processes during bioproduction, serve as environmental sentinels or guide living therapeutics.

A Computational Workflow for the Automated Generation of Models of Genetic Designs.

Computational models are essential to engineer predictable biological systems and to scale up this process for complex systems. Computational modeling often requires expert knowledge and data to build models. Clearly, manual creation of models is not scalable for large designs. Despite several automated model construction approaches, computational methodologies to bridge knowledge in design repositories and the process of creating computational models have still not been established. This paper describes a workflow for automatic generation of computational models of genetic circuits from data stored in design repositories using existing standards. This workflow leverages the software tool SBOLDesigner to build structural models that are then enriched by the Virtual Parts Repository API using Systems Biology Open Language (SBOL) data fetched from the SynBioHub design repository. The iBioSim software tool is then utilized to convert this SBOL description into a computational model encoded using the Systems Biology Markup Language (SBML). Finally, this SBML model can be simulated using a variety of methods. This workflow provides synthetic biologists with easy to use tools to create predictable biological systems, hiding away the complexity of building computational models. This approach can further be incorporated into other computational workflows for design automation.