I am now working on a complementation experiment of a deleted gene with the TrpC promoter and terminator on hygromycin cassette, but it doesn't work well because the phenotype is complementary, it can't grow on the PDA with hygromycin. I suspect the TrpC promoter may not work well. It is that a problem?
Dear Dianguang,
The expression level given by the promoters of TrpC and gpdA might be different, but both are known to be very strong promoters in Ascomycetes. It is unlikely that replacing the promoter of TrpC with the one of gpdA in front of your selectable marker will solve your problem.
It seems more probable to me that you are having a problem with your transformation procedure, or with the selection of your transformants. If I have understood your message properly, you said that your "recombinant" colonies failed to grow on selective medium. I assumed that you could recover colonies from your primary selection plates, but that they did not grow after you picked them on a new batch of selective medium. It is a quite common problem with the transformation of fungi. These colonies might be false positive, and harbor neither the gene that your are working on, nor the selectable marker. In this case, your transformation experiment has simply failed. These colonies might also contain a mixture of recombinant and wild-type nuclei, especially if you are working with a fungus made of cells having more than one nucleus. In this case, if you do not keep constant selection pressure and if you do not "purify" the recombinant nuclei (usually by single spore isolation), you may "lose" the selectable marker. That is another possible explanation for what you observed.
You did not precise in your question which is the fungus you are working on and what is the transformation procedure you used, so it is difficult for me to say more, but I hope it will help.
Pierre-Henri
Thank you very much. It really useful for me. I am now working on the Verticillium dahliae. Actually i am not keep a constant selection pressure, maybe that's the problem and I will correct it. Thank you.
I am glad it helped. I have never been working with V. dahliea myself, but several labs in the world are. Protocols of transformation must be available on request or in publications. I am advising you to follow their selection procedure carefully. This is the best way to start solving your problem.
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