I am trying to introduce a DNA fragment for recombinant protein expression in Trichoderma spp by using electroporation but there are no transformants (using linearized plasmid with hygromycin B resistance under the control of a constitutive promoter and cel7a promoter controles expression of a non toxic protein directed for exportation). I am using fresh conidiospores and the electroporation conditions reported in the literature. Could someone help me with some trick to solve this?
Thank you in advance,
Do you allow the spores to express the hygromycine marker, before applying the selection?
After electroporation we keep the spores for 2 h in 1M sobitol solution. In your experience is this enough? Prior to electroporation spores were incubated for 8 h in YPD to activate as it takes 8-10 h to germinate.
Thank you for your suggestion.
It might help incubating the "zapped" spores several hours or overnight in YPD before plating them on selective plates. This would allow proper expression of the hph gene.
I did a lot of elettroporation with T. reesei. It's working fine, but you need a high quality DNA and in big amount (10µg por cuvette). one more trick is to collect spores from 7-12 days old plates (no older). I wish it helps you
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