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

被引：134

作者：

Beyer, Hannes M. ^{[1
,2
]}

Gonschorek, Patrick ^{[1
]}

Samodelov, Sophia L. ^{[1
,2
]}

Meier, Matthias ^{[3
,4
]}

Weber, Wilfried ^{[1
,2
,3
]}

Zurbriggen, Matias D. ^{[1
,3
]}

机构：

[1] Univ Freiburg, Fac Biol, D-79106 Freiburg, Germany

[2] Univ Freiburg, Spemann Grad Sch Biol & Med SGBM, D-79106 Freiburg, Germany

[3] Univ Freiburg, BIOSS Ctr Biol Signalling Studies, D-79106 Freiburg, Germany

[4] Univ Freiburg, Dept Microsyst Engn, IMTEK, D-79106 Freiburg, Germany

来源：

PLOS ONE | 2015年 / 10卷 / 09期

基金：

欧洲研究理事会;

关键词：

SYNTHETIC BIOLOGY; CONSTRUCTION; EXPRESSION;

D O I：

10.1371/journal.pone.0137652

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页数：20

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