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 one-step protocol employs efficient single-stranded DNA annealing proteins (SSAP) to incorporate (libraries of) DNA oligos harboring the desired mutations into a target plasmid in E. coli. MOSAIC can be used to modify virtually any plasmid and is integrated with a validation pipeline based on Nanopore sequencing. In addition to up to 90% single-target plasmid editing efficiency, MOSAIC is demonstrated to enable the generation of a combinatorial plasmid library spanning four different target regions on a plasmid, in a single transformation. 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵† Joint Authors
https://dx.doi.org/10.4121/4464ab86-9214-49b3-a808-10ca655385a6