Reflections on DNA Assembly Practice

Metabolic engineering presents the possibility of creating novel and practical whole-cell biocatalysts. The practice of metabolic engineering is achieved first by in vitro DNA assembly, followed by the introduction of the newly constructed DNA into industrial microorganisms to create a novel phenotype. Although this approach of in vitro DNA assembly has been studied extensively, generation of unwanted recombinant DNA products remains a possibility. In this study, a recombinant DNA, namely pGRN02, was constructed using the sequence- and ligation- independent cloning. However, this DNA assembly method had a low success rate (5%). Unexpectedly, we identified an un-wanted recombinant DNA product as a major recombinant product (70%). DNA sequencing of this product indicated that it should not have been formed during in vitro DNA assembly, but rather post in vitro assembly. This study aims to report and discuss profound results of the DNA assembly reaction. The standard SLIC design using 20 bp homology arms is theoretically sufficient for correct assembly under typical conditions. However, longer unexpected repeats, such as the 44 bp internal homology observed here, can outcompete the designed junctions and dominate the recombination outcome.