An att site-based recombination reporter system for genome engineering and synthetic DNA assembly.

Michael J Bland
Michael J Bland
Valdosta State University
United States
Didier Mazel
Didier Mazel
Institut Pasteur
Paris | France

BMC Biotechnol 2017 Jul 14;17(1):62. Epub 2017 Jul 14.

Unité Plasticité du Génome Bactérien, Département Génomes et Génétique, Institut Pasteur, 75015, Paris, France.

Background: Direct manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications. The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB site from each system within the β-lactamase resistance coding sequence (bla).

Results: The HK and ΦC31 tools were developed by placing the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs (3 for HK, 6 for ΦC31) as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested.

Conclusions: The HK/ΦC31 att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.

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