Abstract

The field of Synthetic Biology has seen many recent developments in DNA assembly protocols, software tools in aide of assembly design and experiment planning, standardization of parts, and automation of complex and high-throughput DNA assembly and cloning processes. These software tools reduce human-introduced errors and the need for highly-skilled labor, cut-down the total cost of assembly experiments, and diminish the overall time expended in designing, planning and executing reliable cloning experiments. Shortcomings of these efforts, however, exist in their usability and accessibility, support for major cloning methods, the incorporation of both part repositories and direct-synthesis DNA sequences in the design process, extensibility of the software, and the lack of a comparison of assembly design options offered by different assembly methods. Here, Smithy is presented: a web-application automating DNA assembly project design for scar-less assemblies, incorporating existing sequences from major repositories (e.g., AddGene and iGEM) and synthetic DNA sequences into the assembly design process. Currently, the supported cloning methods are Gibson, Golden Gate, PCR-SOE, SLIC and BioBricks. Smithy software architecture utilizes a new DNA cloning methods abstraction hierarchy, which facilitates effective and extendible application implementation and extendibility. Smithy’s graphical user interface and overall user experience are simpler and more informative than existing DNA assembly design tools. As a service, Smithy is open-source, free, and widely accessible for users of all experience levels. The core software features exist as independent modules that are portable to future automation projects. Furthermore, an analytical feature for creating assembly bundles, has been developed. Assembly bundles comprise of multiple assembly solutions for the same target construct; this facilitates quick comparison and selection of the most appropriate assembly solution by and for a particular user. To exemplify the usefulness of Smithy, two detailed case studies are presented: a single Gibson assembly for a riboswitch-modulated fluorescent genetic circuit, and another bundle assembly for an expanded riboswitch-modulated fluorescent genetic circuit, employing Gibson, Golden Gate and PCR-SOE methods. Finally, Smithy is a highly usable, future-oriented tool, which can be improved and expanded in response to future developments in cloning methodologies, standardization, and software tools, in the highly promising field of Synthetic Biology.