I am working on a project where I need to delete an essential gene (null mutation), while keeping a duplicate (with alterations) on a plasmid.
However I was wondering if it is easier to make a gene disruption of the essential gene with a URA marker, in a haploid strain with the URA gene deleted in Uracil containing medium. After the transformation of the strain with a KanMX marker and then selecting on Uracil-free plates containing G418.
Inputs are much appreciated.
Best regards
Chris S. Ramming
I do not understand how your proposed strategy could make it easier or make it work. I did essential gene deletion and analysis different mutation effect of the gene in the past with haploid strain. I could not say it was easy but It served my purpose. Here were how I did: 1, transform a duplicate of the gene with URA3 marked plasmid. 2, delete the genome copy of the gene by replacement with KanMX. 3, transform with designed mutated gene with Leu2 selection. 4, drive out the wt gene-URA3 plasmid by 5FOA. If the wt gene-URAs cannot be drived out, the mutant destroys the normal function of the gene. The strain of wt gene URA3 drived out may be normal or show some phenotypes reflecting your designed mutation effect. Hope that helps.
Sorry i did not elaborate this to its full extend. but you explained a similar approach as i was thinking. But thank you very much for the great answer.
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