| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2091596 | Journal of Microbiological Methods | 2009 | 6 Pages |
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.
