Integrases on Demand

All Authors

Jesse Cahill, SNL Lauren Clark, NU Greyson Lasley, SNL Brittany Humphrey, SNL Mia Lobitz, SNL Catherine Mageeney, SNL Michael Jewett, NU Joe Schoeniger, SNL Kelly Williams, SNL

During infection of the host cell, phages produce integrases and excisionases to insert or remove phage DNA at specific attachment (att) sites in the host chromosome. Of the two integrase protein families, serine integrases can accomplish these DNA rearrangements in the absence of other proteins, so they are widely used in genome engineering of highly diverse organisms. However, the more abundant tyrosine integrases are reported to require integration host factor (IHF) for function, limiting their biotech use. Integrase site-specificity also limits genome editing capabilities when the genome lacks a compatible att site. Therefore, a library of integrases/cognate att sites would revolutionize genomic engineering. Our team developed software (Islander/TIGER) and scanned ~400,000 genomes, allowing us to identify ~1 million instances of integrases paired with their att sites. To functionally validate a subset of the predicted integrases, we developed both a cell-free and an in vivo testbed. For the in vivo assay, each test att site pair was arranged so that the integrase would invert the lacZ_ gene in E. coli. When screened on appropriate media, colonies carrying functional integrases appear blue. Out of 45 total tested, 57% (26) are functional and 21 of these were tyrosine integrases. Unexpectedly, many tyrosine integrases from diverse phyla outside that of E. coli (e.g. cyanobacteria) function in E. coli. This finding challenges the notion of strict IHF requirement. Two simple explanations are that some tyrosine integrases are IHF independent or the E. coli IHF is sufficient to support diverse tyrosine integrases. We are currently investigating these possibilities.