The T. reesei Δtku70 strain [20 (link)] was used for transformation in order to achieve a high efficiency in homologous integration of the deletion cassettes. The protoplast transformation was carried out as previously described [13 (link)]. For transformation 2.5 μg of purified deletion cassette fragment was used. Transformants were grown on selective minimal medium (1 g/liter MgSO4*7H2O, 10 g/liter 1% KH2PO4, 6 g/liter (NH4)2SO4, 3 g/liter trisodium citrate*2H2O, 10 g/liter glucose, 20 ml/liter 50x trace elements solution (0.25 g/liter FeSO4*7H2O, 0.07 g/liter ZnSO4*2H2O, 0.1 g/liter CoCl2*6H2O, 0.085 g/liter MnSO4*H2O), 2% (wt/vol) agar; all chemicals were from Sigma Aldrich, St. Louis, USA).
The protocol for electroporation of T. reesei was adapted from patent application US2010/0304468. Spores of T. reesei Δtku70 were harvested from a freshly sporulated 90 mm malt extract agar plate and suspended in 1.1 M sorbitol. Spores were washed twice, resuspended in 100 μl 1.1 M sorbitol and cooled on ice. 75 μl of cold spore suspension was mixed with 10 μg of vector of interest. For electroporation we used an Electroporation System ECM® 630 (BTX Instrument Division Harvard Apparatus, Holliston, USA). 1.8 kV, 800 Ω and 25 μF were used as setting for the ECM® 630 device. Thereafter one volume of the reaction mixture (consisting of spore suspension plus deletion cassette) of YEPD (1% (w/v) yeast extract, 2% (w/v) peptone, 1% (w/v) glucose) and four reaction volumes 1.1 M sorbitol were added and mixed. For regeneration the whole mixture was incubated over night at room temperature. Thereafter, spores were streaked out on plates containing selection medium.
Compared to the classical protoplast transformation technique [13 (link)] transformation by electroporation is less time consuming, easier to perform and the efficiency of this method was comparable to that of protoplast transformation.
Putative deletion strains were tested for integration of the construct by PCR using primer pyr4Sc (inside in the selectable marker gene pyr4) and the gene-specific primer, geneSc (outside from the transformation cassette) (supplementary file 1). Successful homologous integration results in a specific amplicon (1400 - 2000 bp's, depending on the gene) (supplementary file 1) and no amplification was possible with T. reesei Δtku70 genomic DNA (data not shown).
The protocol for electroporation of T. reesei was adapted from patent application US2010/0304468. Spores of T. reesei Δtku70 were harvested from a freshly sporulated 90 mm malt extract agar plate and suspended in 1.1 M sorbitol. Spores were washed twice, resuspended in 100 μl 1.1 M sorbitol and cooled on ice. 75 μl of cold spore suspension was mixed with 10 μg of vector of interest. For electroporation we used an Electroporation System ECM® 630 (BTX Instrument Division Harvard Apparatus, Holliston, USA). 1.8 kV, 800 Ω and 25 μF were used as setting for the ECM® 630 device. Thereafter one volume of the reaction mixture (consisting of spore suspension plus deletion cassette) of YEPD (1% (w/v) yeast extract, 2% (w/v) peptone, 1% (w/v) glucose) and four reaction volumes 1.1 M sorbitol were added and mixed. For regeneration the whole mixture was incubated over night at room temperature. Thereafter, spores were streaked out on plates containing selection medium.
Compared to the classical protoplast transformation technique [13 (link)] transformation by electroporation is less time consuming, easier to perform and the efficiency of this method was comparable to that of protoplast transformation.
Putative deletion strains were tested for integration of the construct by PCR using primer pyr4Sc (inside in the selectable marker gene pyr4) and the gene-specific primer, geneSc (outside from the transformation cassette) (supplementary file 1). Successful homologous integration results in a specific amplicon (1400 - 2000 bp's, depending on the gene) (supplementary file 1) and no amplification was possible with T. reesei Δtku70 genomic DNA (data not shown).
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