Post-assembly Plasmid\nAmplification for Increased\nTransformation Yields in <i>E. coli</i> and <i>S. cerevisiae</i>

Many biological disciplines rely upon the transformation\nof host\ncells with heterologous DNA to edit, engineer, or examine biological\nphenotypes. Transformation of model cell strains (<i>Escherichia\ncoli</i>) under model conditions (electroporation of circular\nsupercoiled plasmid DNA; typically pUC19) can achieve >10<sup>10</sup> transformants/μg DNA. Yet outside of these conditions, e.g.,\nwork with relaxed plasmid DNA from <i>in vitro</i> assembly\nreactions (cloned DNA) or nonmodel organisms, the efficiency of transformation\ncan drop by multiple orders of magnitude. Overcoming these inefficiencies\nrequires cost- and time-intensive processes, such as generating large\nquantities of appropriately formatted input DNA or transforming many\naliquots of cells in parallel. We sought to simplify the generation\nof large quantities of appropriately formatted input cloned DNA by\nusing rolling circle amplification (RCA) and treatment with specific\nendonucleases to generate an efficiently transformable linear DNA\nproduct for <i>in vivo</i> circularization in host cells.\nWe achieved an over 6500-fold increase in the yield of input DNA,\nand demonstrate that the use of a nicking endonuclease to generate\nhomologous single-stranded ends increases the efficiency of <i>E. coli</i> chemical transformation compared to both linear\nDNA with double-stranded homologous ends and circular Golden-Gate\nassembly products. Meanwhile, the use of a restriction endonuclease\nto generate linear DNA with double-stranded homologous ends increases\nthe efficiency of chemical and electrotransformation of <i>Saccharomyces\ncerevisiae</i>. Importantly, we also optimized the process such\nthat both RCA and endonuclease treatment occur efficiently in the\nsame buffer, streamlining the workflow and reducing product loss through\npurification steps. We expect that our approach could have utility\nbeyond <i>E. coli</i> and <i>S. cerevisiae</i> and be applicable to areas such as directed evolution, genome engineering,\nand the manipulation of alternative organisms with even poorer transformation\nefficiencies.