Journal: Nature Communications

Article Title: PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction

doi: 10.1038/s41467-022-30355-y

Figure Lengend Snippet: Design, Build, Test cycle for construction of plasmids from linear DNA parts. In the first step, a frontend is used to design and choose DNA fragments for assembly. After all the selected sequences passed the quality check, picklists (input for “Build” part) are generated for large-scale synthesis of primers and guides. 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. Next, one-pot digestion, ligation, and transformation is performed inside iBioFAB to assemble DNA fragments. Pf Ago-based AREs are used for DNA assembly. In the first step, linear DNA molecules ends are digested with WT and engineered Pf Ago/AREs in a one-pot reaction. The AREs generate 5′ sticky ends of 12 nt length. After purification, the digested DNA molecules are assembled in vitro using a high-fidelity DNA ligase and assembly products are transformed into E. coli cells for screening. Finally, constructed plasmids (input for “Test” part) are checked and the correct plasmids are stocked using the robotic system.

Article Snippet: DNA purification buffers PB and PE were purchased from Qiagen, Germany.

Techniques: Generated, Ligation, Transformation Assay, Purification, In Vitro, Construct