Post-assembly Plasmid Amplification for Increased Transformation Yields in E. coli and S. cerevisiae.

Abstract

Many biological disciplines rely upon the transformation of host cells with heterologous DNA to edit, engineer, or examine biological phenotypes. Transformation of model cell strains (Escherichia coli) under model conditions (electroporation of circular supercoiled plasmid DNA; typically pUC19) can achieve >10¹⁰ transformants/μg DNA. Yet outside of these conditions, e.g., work with relaxed plasmid DNA from in vitro assembly reactions (cloned DNA) or nonmodel organisms, the efficiency of transformation can drop by multiple orders of magnitude. Overcoming these inefficiencies requires cost- and time-intensive processes, such as generating large quantities of appropriately formatted input DNA or transforming many aliquots of cells in parallel. We sought to simplify the generation of large quantities of appropriately formatted input cloned DNA by using rolling circle amplification (RCA) and treatment with specific endonucleases to generate an efficiently transformable linear DNA product for in vivo circularization in host cells. We achieved an over 6500-fold increase in the yield of input DNA, and demonstrate that the use of a nicking endonuclease to generate homologous single-stranded ends increases the efficiency of E. coli chemical transformation compared to both linear DNA with double-stranded homologous ends and circular Golden-Gate assembly products. Meanwhile, the use of a restriction endonuclease to generate linear DNA with double-stranded homologous ends increases the efficiency of chemical and electrotransformation of Saccharomyces cerevisiae. Importantly, we also optimized 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 that our approach could have utility beyond E. coli and S. cerevisiae and be applicable to areas such as directed evolution, genome engineering, and the manipulation of alternative organisms with even poorer transformation efficiencies.