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

Beyer et al. (2015). PLoS ONE DOI: 10.1371/journal.pone.0137652 Citations: 217

Seamless DNA assembly still relied on specific enzymes and kits. AQUA cloning joins 16-32 bp overlapping fragments via E. coli's native recombination, no enzymes needed. It assembled fragments in one pot into a light- and chemically regulated logic-gate plasmid.

Parts used

mCherryEGFPIRESSV40 NLSNESP2A Self-Cleaving Peptide

Key findings

  • The AQUA cloning method was developed using homologous recombination in Escherichia coli with 16-32 bp overlaps between DNA fragments. Parameter screening of 2-fragment assemblies revealed optimal conditions at room temperature with 32 bp homology using PCR-derived DNA, yielding 1,980 CFU with highest accuracy. The method showed broad compatibility across E. coli strains (TOP10, NEB5α, NEB10β, BL21(DE3), JM109) with 83-100% assembly accuracy, and required no restriction enzymes, ligases, or exonucleases.

  • AQUA cloning enabled diverse molecular biology applications including site-directed mutagenesis (Y66H substitution in EGFP with blue-shifted fluorescence), insertion/deletion mutagenesis (Lck membrane anchor deletion and MTS insertion for subcellular targeting), and combined cloning-expression (AQUA Expression) in BL21(DE3) that produced recombinant mCherry protein within 24 hours from PCR to purification. The method tolerated variations in homology length (16-32 bp) and showed low vector self-ligation (1.06%).

  • A 6-fragment de novo assembly constructed a bicistronic mammalian expression plasmid encoding a light- and chemically-regulated A NIMPLY B Boolean logic gate using UVR8-TetR and COP1-VP16 fusions separated by IRES. Functional validation in CHO-K1 cells demonstrated UV-B light-dependent SEAP reporter expression that was dominantly inhibited by tetracycline, confirming correct assembly and logical output. Additional 4-fragment assemblies (mCherry-NLS/IRES/EGFP-NES) and combinatorial plant auxin sensors showed 83% assembly accuracy.

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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.