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bioRxiv · 10.1101/2024.03.22.586135

MOSAIC: a highly efficient, one-step recombineering approach to plasmid editing and diversification

van den Brink, M.·Althuis, T. Y.·Danelon, C.·Claassens, N. J.
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Abstract

The editing of plasmids and construction of plasmid libraries is paramount to the engineering of desired functionalities in synthetic biology. Typically, plasmids with targeted mutations are produced through time- and resource-consuming DNA amplification and/or cloning steps. In this study, we establish MOSAIC, a highly efficient protocol for the editing of plasmids and generation of combinatorial plasmid libraries. This quick protocol employs the efficient single-stranded DNA annealing protein (SSAP) CspRecT to incorporate (libraries of) DNA oligos harboring the desired mutations into a target plasmid in E. coli. In addition to up to 90% single-target plasmid editing efficiency, we demonstrate that MOSAIC enables the generation of a combinatorial plasmid library spanning four different target regions on a plasmid, in a single transformation. Lastly, we integrated a user-friendly validation pipeline using Nanopore sequencing reads, requiring minimal computational experience. We anticipate that MOSAIC will provide researchers with a simple, rapid and resource-effective method to edit plasmids or generate large, diverse plasmid libraries for a wide range of in vivo or in vitro applications in molecular and synthetic biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/586135v3_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@929537org.highwire.dtl.DTLVardef@1825039org.highwire.dtl.DTLVardef@60e89aorg.highwire.dtl.DTLVardef@46ef46_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

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van den Brink, M., Althuis, T. Y., Danelon, C., Claassens, N. J.. 2024-03-22. MOSAIC: a highly efficient, one-step recombineering approach to plasmid editing and diversification. https://doi.org/10.1101/2024.03.22.586135

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