A Golden Gate-based efficient assembly system of transcription units for Sclerotium rolfsii

Abstract:

[Objective] To establish an efficient transcription unit assembly system for the metabolic engineering of Sclerotium rolfsii. [Methods] Based on Golden Gate technology and Mobius assembly, the scaffold plasmids for DNA part domestication, single transcription unit assembly, and application plasmid (multi-transcription-unit) assembly, were designed and constructed to provide a complete multi-transcription-unit assembly system. [Results] Two Level 0 vectors for DNA part domestication, four Level 1 vectors for single transcription unit assembly, and four Level 2 vectors and 13 auxiliary plasmids for application plasmid assembly were constructed. Using this system, we assembled tens of plasmids for domesticated DNA parts, single transcription units, and function analysis of scleroglucan-related genes. The constructed application plasmids could be directly applied in Agrobacterium tumefaciens-mediated transformation, electroporation transformation, and protoplast transformation of S. rolfsii. [Conclusion] The plasmid system we built has strong capabilities of DNA design, assembly, and capacity, providing an efficient plasmid construction platform for metabolic engineering and functional genomics research of S. rolfsii in the future.