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

• Published in: PloS one Vol. 10; no. 9; p. e0137652
• Main Authors: Beyer, Hannes M, Gonschorek, Patrick, Samodelov, Sophia L, Meier, Matthias, Weber, Wilfried, Zurbriggen, Matias D
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 11.09.2015; Public Library of Science (PLoS)
• Subjects: Analysis; Assembly; Boolean algebra; Clonal deletion; Cloning; Cloning, Molecular - methods; Combinatorial analysis; Deoxyribonucleic acid; Distribution; DNA; DNA polymerase; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fragments; Gene deletion; Gene Expression; Genetic aspects; Genetic engineering; Harnesses; Homology; Insertion; Mutagenesis; Mutagenesis, Site-Directed; Plasmids; Plasmids - genetics; Reagents; Studies; Synthetic biology; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0137652

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.