کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8420311 | 1545893 | 2018 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Targeted cloning of a large gene cluster from Lecanicillium genome by Cre/loxP based method
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیوتکنولوژی یا زیستفناوری
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چکیده انگلیسی
Functionally related genes often form a large gene cluster on fungal genomes. To analyze, by heterologous expression system, overall pathway in which a series of related genes are involved, the whole gene cluster should be introduced intact into the host strain. However, the construction of a genomic library based on cosmid or bacterial artificial chromosome, and screening of a clone harboring the target region are time consuming and usually require additional cloning of missing regions. The available PCR-based methods are convenient, but are likely to cause unexpected errors during long-range PCR. Therefore, in this study we developed a method for targeted cloning of a large gene cluster based on Cre/loxP-mediated recombination. loxP sequences were integrated at both edges of the targeted region, and the region was excised and cloned as a circular fosmid by in vitro Cre recombination. To facilitate the Cre/loxP-based method, a competent host-vector system was developed, including a double auxotrophic Lecanicillium PTk3 (ÎpyrG trp1âku80â) strain and two vectors for introducing the loxP sequences, pUTlox and pCCPlox. A targeted region longer than 45â¯kb in length was successfully cloned by the Cre/loxP-based method.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Microbiological Methods - Volume 150, July 2018, Pages 47-54
Journal: Journal of Microbiological Methods - Volume 150, July 2018, Pages 47-54
نویسندگان
Havy N. Nguyen, Kei-ichi Ishidoh, Hiroshi Kinoshita, Takuya Nihira,