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Abstract
BackgroundDirect 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).ResultsThe 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.ConclusionsThe 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.
Highlights
Direct manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications
In this study, we describe the construction of two sitespecific recombination tools useful for DNA manipulation applications
In directly comparing the two systems, the ΦC31 site appears to recombine at a similar frequency to the 51 bp HK sites and the 23 bp HK Open reading frame (ORF) incorporated into bla have a lower recombination frequency (Fig. 2)
Summary
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. Site-specific recombinase (SSR) systems are widely used as tools to rearrange, insert, remove, and join DNA with virtually no upper limit in size. For biotechnology purposes, this can include the insertion of exogenous DNA into chromosomes, the fusing of DNA molecules, or the construction of synthetic gene networks [1]. Important members of the Y-rec family include the λ-like phage recombination systems, Mechanistically, attB and attP integrative recombination forms attL and attR sites.
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