PlayBack cloning: simple, reversible, cost-effective cloning for the combinatorial assembly of complex expression constructs

With advancements in multicomponent molecular biological tools, the need for versatile, rapid and cost-effective cloning that enables successful combinatorial assembly of DNA plasmids of interest is becoming increasingly important. Unfortunately, current cloning platforms fall short regarding affordability, ease of combinatorial assembly and, above all, the ability to iteratively remove individual cassettes at will. Herein we construct, implement and make available a broad set of cloning vectors, called PlayBack vectors, that allow for the expression of several different constructs simultaneously under separate promoters. Overall, this system is substantially cheaper than other multicomponent cloning systems, has usability for a wide breadth of experimental paradigms and includes the novel feature of being able to selectively remove components of interest at will at any stage of the cloning platform. PlayBack cloning offers a versatile, rapid, inexpensive platform for the combination of multiple DNA constructs into a single plasmid vector with the unique feature of selective cassette removal at any stage of plasmid DNA cloning. Herein we introduce PlayBack cloning, a platform designed to allow complex combinatorial assembly of multiple DNA constructs into a single plasmid vector with the ability to selectively add/remove any component of interest at any stage of assembly. PlayBack cloning relies upon the insertion of matching restriction enzyme sites that flank each cassette of interest, thereby allowing for the selective and iterative removal of individual cassettes of interest from a multi-construct-containing plasmid, which can then be self-ligated, allowing its direct use as an expression vector or as a platform for subsequent genetic modification.

Automated summary

This study introduces PlayBack cloning, a cloning platform designed to allow rapid and inexpensive combination of multiple DNA constructs into a single plasmid vector, with the ability to selectively add or remove components of interest at any stage of assembly.