کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2091596 1081740 2009 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Improved electroporation-mediated non-integrative transformation of Thermomyces lanuginosus
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
Improved electroporation-mediated non-integrative transformation of Thermomyces lanuginosus
چکیده انگلیسی

The development of an efficient fungal expression system for recombinant proteins requires an improved transformation system for the host organism. We report a facile, efficient and highly reproducible electroporation-mediated transformation system for Thermomyces lanuginosus with a transformation efficiency of 1.27 × 103 transformants/µg DNA. Conidia of T. lanuginosus were stably transformed to hygromycin B resistance using the pBC-hygro plasmid construct. Optimal electroporation conditions for maximum transformation of 108 conidia ml− 1 in 1.2 M sorbitol buffer (15 mM DTT, 5% DMSO) were a field strength of 5.5 kV/cm for 10 ms and a DNA concentration of 0.5 µg µl− 1. Transformants were recovered in prewarmed potato dextrose broth supplemented with 1.2 M sorbitol for 1–2 h at 50 °C. The presence of the hygromycin B phosphotransferase (hph) gene and non-integrative transformation was confirmed by PCR, Southern hybridization analysis and plasmid recovery. Transformants exhibited altered phenotype with reduced pigmentation and transformants were found to be mitotically stable after 15 sequential transfers on nonselective media without selective pressure.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Microbiological Methods - Volume 77, Issue 2, May 2009, Pages 159–164
نویسندگان
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