Most of the GFP-ATG8 studies use yeast-derived plasmids rather than integrating into the genome. For the ones that integrate GFP-ATG8 into the genome, they are using a commercially available URA3 marker for the URA3 locus, which is not available in BY4741. Is there any other way than making a new plasmid? Thank you!
Hello Songlin Wu
GFP tagged Atg8 is stable when it is tagged at the N-terminal. You can prepare a PCR cassette with Atg8 N- and C-terminal homology following GFP sequence in frame. For the selection of the right transformation, you need a selection marker which can be either an auxotrophic marker (for yeast strain) or an antibiotic marker (Kanamycin/Nourseothricin). BY4741 strain has uracil auxotrophy (ura3Δ 0). This URA3 gene will be integrated first at the 5'-UTR of the gene (between promoter and GFP sequence), but then you have to eliminate it using Cre-recombinase via loxP sites.
Best wishes.
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