Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiaeReportar como inadecuado




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BMC Biotechnology

, 16:13

Microbial biotechnology

Abstract

BackgroundPhage-encoded serine integrases, such as ϕC31 integrase, are widely used for genome engineering but have not been optimized for use in Saccharomyces cerevisiae although this organism is a widely used organism in biotechnology.

ResultsThe activities of derivatives of fourteen serine integrases that either possess or lack a nuclear localization signal were compared using a standardized recombinase mediated cassette exchange reaction. The relative activities of these integrases in S. cerevisiae and in mammalian cells suggested that the major determinant of the activity of an integrase is the enzyme itself and not the cell in which it is working. We used an inducible promoter to show that six integrases were toxic as judged by their effects upon the proliferative ability of transformed yeast. We show that in general the active phage-encoded serine integrases were an order of magnitude more efficient in promoting genome integration reactions than a simple homologous recombination.

ConclusionsThe results of our study allow us to identify the integrases of the phage ϕBT1, TP901 ~ nls, R4, Bxb1, MR11, A118, ϕK38, ϕC31 ~ nls, Wβ and SPBC ~ nls as active in S. cerevisiae and indicate that vertebrate cells are more restricted than yeast in terms of which integrases are active.

KeywordsPhage encoded serine integrases Cassette exchange Saccharomyces cerevisiae Genome modification AbbreviationsNLSNuclear localization signal

Electronic supplementary materialThe online version of this article doi:10.1186-s12896-016-0241-5 contains supplementary material, which is available to authorized users.

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Autor: Zhengyao Xu - William R. A. Brown

Fuente: https://link.springer.com/







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