Hello everyone. I have a question related to gene integration in the genome of Saccharomyces cerevisiae using a plasmid. Can anyone help me with the gene integration procedure in yeast using the plasmid?
Hi Rudrakshi Sharma
For genomic integration, you can use different type of plasmids: integrative plasmids like YIplac128 (LEU2 auxotrophy marker) and YIplac211 (URA3 auxotrophy marker) are ideal for the genomic integration. You need to clone the gene, ligate into the plasmid with identical restriction sites followed by linearization of the plasmid in the inserted gene sequence and transformation. These are the most effective transformation.
If the genes are too long or difficult to clone, you can use PCR cassette such as pKT series plasmid e.g. pKT128 (HIS3 auxotrophy selection).
You can also use pFA6a cassette, these have both antibiotic selection markers (kanamycin and nourseothricin) and auxotrophic markers.
I am hereby attaching some of the articles regarding yeast plasmids for your better understanding.
Best wishes.
Hi there,
Integration occurs via homologous recombination in yeast. To optimize this integration you have to linearize the plasmid within the region where recombination events are expected so that extremities of your linearized plasmid are homologous to the site of insertion. The plasmid has to bear a selection marker (as linearized plasmid is not stable in the cell, selection will be made on insertion).
Dominique is right. I just want to ask which plasmid you have. Which marker does it carry? Which yeast strain do you want to use. Please report its genotype. BEWARE: Not every marker goes in every yeast strain, some combinations do not work as you need homologous recombination.
Hey J. Stolz , Dominique Liger, I am using a modified plasmid of the pYAC vector. It has the His marker and I will be using the CCFY101 strain for the process. Need the valuable input for the same.
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