After purification, the digested DNA molecules are assembled in vitro using a high-fidelity DNA ligase and assembly products are transformed into E. The AREs generate 5′ sticky ends of 12 nt length. In the first step, linear DNA molecules ends are digested with WT and engineered PfAgo/AREs in a one-pot reaction. PfAgo-based AREs are used for DNA assembly. Next, one-pot digestion, ligation, and transformation is performed inside iBioFAB to assemble DNA fragments. The received oligos in either 96-well format or 384-well format are transferred into corresponding destination wells for PCR reactions to make specific DNA fragments via liquid handlers. After all the selected sequences passed the quality check, picklists (input for “Build” part) are generated for large-scale synthesis of primers and guides. In the first step, a frontend is used to design and choose DNA fragments for assembly. Here the authors report a robust, versatile, and automated end-to-end platform that enables scarless construction of virtually any plasmid.ĭesign, Build, Test cycle for construction of plasmids from linear DNA parts. Despite their broad utility, design and construction of plasmids remains laborious and time-consuming. 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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