title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach.
abstract: 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.
authors.affiliation: Faculty of Biology, University of Freiburg, Freiburg, Germany; Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany.
authors.forename: Hannes M
authors.initials: HM
authors.lastname: Beyer
authors.affiliation: Faculty of Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Gonschorek
authors.affiliation: Faculty of Biology, University of Freiburg, Freiburg, Germany; Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany.
authors.forename: Sophia L
authors.initials: SL
authors.lastname: Samodelov
authors.affiliation: BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany; IMTEK, Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Meier
authors.affiliation: Faculty of Biology, University of Freiburg, Freiburg, Germany; Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Weber
authors.affiliation: Faculty of Biology, University of Freiburg, Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Matias D
authors.initials: MD
authors.lastname: Zurbriggen
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics