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