I am preparing to do a gene knockout in the filamentous fungi Coccidioides posadasii targeting pyrG/URA3 so that 5-FOA can be used for selection pressure instead of traditional antibiotic selection pressure.

I am using a protoplasting/transformation protocol that was developed using hygromycin as a selection pressure, which I will be substituting with 5-FOA. This protocol calls for a sort of "agar sandwich" where the transformed protoplasts are covered in soft agar without antibiotics so that they can regenerate, then soft agar with a high concentration of hygromycin is poured on top and diffuses through the media.

I am unsure if 5-FOA will diffuse through the media in the same way as hygromycin. What do you think? I have not been able to find any literature describing this protocol using 5-FOA. Can you think of any other way to apply selection pressure with 5-FOA after allowing the protoplasts some time to regenerate?

Any advice would be much appreciated.

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