I want to design an experiment to confirm that my S. cerevisiae URA3 gene can function in S. cerevisiae.
My plasmid that is a BluescriptiiSK+ harboring S. cerevisiae URA3 gene (and don't has any ARS or 2 micro ori or other yeast gene or sequence). Since, This plasmid have a homolog sequence of Sc URA3 gene so I think maybe it can integrate into yeast genome.
The problem is I cannot find the restriction site that cut only one point on URA3 gene. So, I have to transform an intact circular plasmid into S. cerevisiae.
1. Is it possible to transform an intact circular plasmid to integrate into yeast genome?
2. What method that I should use for transformation? LiAc?
3. What is the quantity (amount) of plasmid that should use for transformation?
or Any suggestions, Please provide.
Best Regrads,
Tatpong
Hi there,
Integration into genome of a circular plasmid is much less efficient than integration of linearized plasmid.
To check the functionality of your URA3 gene why don't you just amplify the URA3 region of your plasmid (or isolate a fragment containing the gene from your plasmid) and transform a ura3- strain with the linear DNA?
Dear Ivanusic,
Thank you for your suggestion.
Yes, please send your protocol to me via this page or my e-mail: [email protected]
Dear Liger,
Thank you for your suggestion.
I can generate fragment of my ScURA3 by restriction enzyme digestion of plasmid. Since my S. cerevisiae strain BY4742 is "ura3 delta 0", Can I check the function of my URA3 gene by transforming this yeast with URA3 fragment? The successful of this method based on homologous recombination (I understand correct or not?). Then, BY4742 that have phenotype like this "ura3 delta 0" can occur homologous recombination? http://www.yeastgenome.org/strain/BY4742/overview
Sorry for my less knowledge about yeast genetic.
If your DNA fragment contains sufficient upstream and downstream sequences of URA3 genomic gene, yes double homologous recombination may occur leading to restoring a genomic copy of URA3 in the delta strain.
Dear Liger,
I already check in a paper, the yeast strain that I used and the URA3 gene on plasmid don't have any homolog sequence (completely deletion of URA3 from this yeast genome). Then, If I still want to test the function of my URA3 gene on my plasmid, Can I transform directly to yeast ura3- strain to check function or I have to move my URA3 gene to the other plasmid that have CEN or ARS to check function.
(I guess the answer is the last?) Thank you
I've checked the ura3 deletion construct from the Brachmann paper (http://onlinelibrary.wiley.com/doi/10.1002/%28SICI%291097-0061%2819980130%2914:2%3C115::AID-YEA204%3E3.0.CO;2-2/epdf), indeed the deletion is more than the ura3 coding sequence (roughly it runs from -220(towards ATG) up to +80(towards stop codon). So if your full gene sequence lies only within this window there is no chance to get homologous recombination.
To answer you latter question (complementation of ura- strain from a stable yeast plasmid), to get positive result you need your gene to be autonomous on the plasmid (efficient yeast promoter and terminator sequences must be located upstream and downstream of the gene coding sequence).
As you expect your ura3 gene to be functional, why don't you just sequence it to compare it with wild type?
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