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.
For a quick check: lay a drop of 5FOA onto an agar and, dry it and locate the actual surface containing 5FOA. Then plate a lawn of a genomic ura+ strain (yeast for instance) and see how the cells are growing. If the effect of 5FOA is occuring outside the original surface then the molecule is diffusing.
Hi there,
If antibiotics diffuse efficiently I don't see why 5FOA (which is even smaller in size) would not... Just have a go!
For a quick check: lay a drop of 5FOA onto an agar rich medium, dry it and locate the actual surface containing 5FOA. Then plate a lawn of a genomic ura+ strain (yeast for instance) and see how the cells are growing. If the effect of 5FOA is occuring outside the original surface then the molecule is diffusing.
For a quick check: lay a drop of 5FOA onto an agar and, dry it and locate the actual surface containing 5FOA. Then plate a lawn of a genomic ura+ strain (yeast for instance) and see how the cells are growing. If the effect of 5FOA is occuring outside the original surface then the molecule is diffusing.
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