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

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...

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Bibliographic Details
Published in:PloS one 2015-09, Vol.10 (9), p.e0137652
Main Authors: Beyer, Hannes M, Gonschorek, Patrick, Samodelov, Sophia L, Meier, Matthias, Weber, Wilfried, Zurbriggen, Matias D
Format: Article
Language:English
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
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0137652