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A new site-specific recombinase-mediated system for targeted multiple genomic deletions employing chimeric loxP and mrpS sites

Abstract

A newly designed site-specific recombination system is presented which allows multiple targeted markerless deletions. The most frequently used tool for removing selection markers or to introduce genes by recombination-mediated cassette exchange is the Cre/loxP system. Many mutant loxP sites have been created for this purpose. However, this study presents a chimeric mutant loxP site denoted mroxP-site. The mroxP site consists of one Cre (loxP/2) and one MrpA (mrpS/2) binding site separated by a palindromic 6-bp spacer sequence. Two mroxP-sites can be recombined by Cre recombinase in head-to-tail as well as in head-to-head orientation. In the head-to-head orientation and the loxP half-sites inside, Cre removes the loxP half-sites during site-specific recombination, creating a new site, mrmrP. The new site is essentially a mrpS site with a palindromic spacer and cannot be used by Cre for recombination anymore. It does, however, present a substrate for the recombinase MrpA. This new system has been successfully applied introducing multiple targeted gene deletions into the Escherichia coli genome. Similar to Cre/loxP and FLP/FRT, this system may be adapted for genetic engineering of other pro- and eukaryotes.

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Acknowledgments

The present work has been completed as part of a PhD thesis at Stuttgart University, Institute of Industrial Genetics (IIG). We would like to thank Prof. Dr. Ralf Mattes for his great and generous support during the past years of research. We further would like to thank Annette Schneck and Silke Weber for technical assistance.

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Correspondence to Josef Altenbuchner.

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Warth, L., Altenbuchner, J. A new site-specific recombinase-mediated system for targeted multiple genomic deletions employing chimeric loxP and mrpS sites. Appl Microbiol Biotechnol 97, 6845–6856 (2013). https://doi.org/10.1007/s00253-013-4827-8

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  • DOI: https://doi.org/10.1007/s00253-013-4827-8

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