AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach

PLoS One. 2015 Sep 11;10(9):e0137652. doi: 10.1371/journal.pone.0137652. eCollection 2015.


Assembly cloning is increasingly replacing conventional restriction enzyme and DNA-ligase-dependent cloning methods for reasons of efficiency and performance. Here, we describe AQUA (advanced quick assembly), a simple and versatile seamless assembly cloning approach. We demonstrate the applicability and versatility of AQUA Cloning in selected proof-of-principle applications including targeted insertion-, deletion- and site-directed point-mutagenesis, and combinatorial cloning. Furthermore, we show the one pot de novo assembly of multiple DNA fragments into a single circular plasmid encoding a complex light- and chemically-regulated Boolean A NIMPLY B logic operation. AQUA Cloning harnesses intrinsic in vivo processing of linear DNA fragments with short regions of homology of 16 to 32 bp mediated by Escherichia coli. It does not require any kits, enzymes or preparations of reagents and is the simplest assembly cloning protocol to date.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular / methods*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Mutagenesis, Site-Directed
  • Plasmids / genetics

Grant support

This work was supported by the Initiating and Networking Fund (IVF) of the Helmholtz Association within the Helmholtz Initiative on Synthetic Biology [SO-078], the European Research Council under the European Community’s Seventh Framework Programme [FP7/2007-2013]/ERC [259043]-CompBioMat, the excellence initiative of the German Federal and State Governments [EXC 294-BIOSS, GSC 4-SGBM], the Alexander von Humboldt Foundation Research Grant No. 1141629, and the German Research Foundation (Emmy-Noether Grant ME3823/1-1).