Some of the most notable examples include sequence homology based methods such as Sequence and Ligation Independent Cloning (SLIC) 3, isothermal Gibson assembly 4, uracil-excision based cloning 5, 6, and yeast homologous recombination 7, 8, or restriction digestion based methods such as MASTER ligation 9 and Golden Gate assembly 10. During the past two decades, a variety of innovative methods for plasmid construction have been developed. Design and construction of plasmids from smaller DNA parts to form complex functional DNA molecules such as biochemical pathways and genetic circuits is essential in molecular biology and represents one of the key steps enabling the design, build, test, and learn (DBTL) cycle in synthetic biology 1, 2. Plasmids are one of the most foundational tools for recombinant DNA technologies. PlasmidMaker should greatly expand the potential of synthetic biology. As a proof of concept, we used this platform to generate 101 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments.
This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute ( PfAgo)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high throughput manner. However, design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. Plasmids are used extensively in basic and applied biology.
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