Simplified post-assembly plasmid library amplification for increased transformation yields inE. coliandS. cerevisiae

Abstract

AbstractMany biological disciplines rely upon the transformation of host cells with heterologous DNA, the limited efficiency of which can significantly hinder a researcher’s work. Directed evolution in particular typically requires the screening of large (thousand-billion member) libraries to identify sequences of interest, and the creation of these libraries at large enough scales to overcome transformation inefficiencies is a cost-and time-intensive process. We simplify this process by using Rolling Circle Amplification (RCA) to amplifyin vitroplasmid DNA assembly reactions, followed by facile resolution of the concatomeric products to monomers through treatment with specific endonucleases and subsequent efficient transformation of the linear DNA products into host cells forin vivocircularisation. We demonstrate that use of a nicking endonuclease to generate homologous single-stranded ends increases the efficiency ofE. colichemical transformation versus both linear DNA with double-stranded homologous ends, and circular golden-gate assembly products, whilst use of a restriction endonuclease to generate linear DNA with double-stranded homologous ends increases the efficiency of chemical and electrotransformation ofS. cerevisiae. Importantly, we also optimise the process such that both RCA and endonuclease treatment occur efficiently in the same buffer, streamlining the workflow and reducing product loss through purification steps. We expect our approach to have utility beyond directed evolution inE. coliandS. cerevisiae, to areas such as genome engineering and the manipulation of alternative organisms with even poorer transformation efficiencies.