(693c) Plasmidmaker: A Versatile, Automated, and High Throughput End-to-End Platform for Plasmid Construction

Plasmids are used extensively in basic and applied biology. 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. We report a robust, versatile, and automated end-to-end platform for plasmid construction named PlasmidMaker that enables scarless and error-free construction of virtually any plasmids in a high throughput manner. To implement this platform, we first developed a versatile, scarless, parallel, robust, and accurate method for assembly of multiple DNA fragments using Pyrococcus furiosus Argonaute (PfAgo) based artificial restriction enzymes (AREs). We then created a user-friendly frontend for plasmid design, and a backend that streamlines the workflow. Finally, we integrated the DNA assembly method and the software with a robotic system named Illinois Biological Foundry for Advanced Biomanufatcuring (iBioFAB) to create a nearly fully automated workflow. As a proof of concept, we used this platform to construct 101 plasmids across 6 different species (bacteria, yeast, plants, and mammals) which involves ~2×10⁴ pipetting steps. Plasmids with sizes ranging from 5 to 18 kb were assembled from up to 11 DNA fragments efficiently with limited human intervention. PlasmidMaker should greatly expand the potential of synthetic biology.