Genes can be integrated into the genome of yeast. This protocol describes how to generate a vector that contains sequences that allow integratioon of any gene into the GAP1 locus of yeast.
Method Article
Gap1 integrative vector
https://doi.org/10.1038/nprot.2007.128
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Genes can be integrated into the genome of yeast. This protocol describes how to generate a vector that contains sequences that allow integratioon of any gene into the GAP1 locus of yeast.
Amplify a pUC fragment containing the bacterial origin of replication and the kanamycin resistance marker by PCR from PCR-BluntII-TOPO (Invitrogen, Carlsbad, CA) and ligate with a PvuII fragment of pDRf1 containing the f1 origin of replication, the PMA1 promoter and ADH3 terminator producing pDL001.
Subclone a synthetic oligonucleotide containing 55 bp upstream of the start (-55 to 0) and 55 bp downstream of the stop (+1810 to 1865) of Gap1 ORF with HpaI and AscI restriction sites into pGEM-T-easy.
Amplify the hphMX3 cassette (for hygromycin B selection in yeast) by PCR from pAG341 and clone into the SpeI site located in the 5’-part of the Gap1 cassette.
Amplify this cassette by PCR and clone into a blunted BglII of pDL001.
Subclone AtAMT1;1, AtAMT1;1-T460A and AtAMT1;1-eGFP from pDR vectors into the KpnI site of the Gap1 integrative plasmid.
Use the yeast strain 31019b (∆::LEU2 ::KanMX2 ura3; see reference 2) to generate DL1 ( Δ Δ) mutant strain and the versions in which AtAMT1;1 or its mutants are integrated.
Transform yeast strain 31019b using a LiAc protocol(3) with an AscI linearized Gap1 integrative vector containing either AtAMT1;1, AtAMT1;1-T460A, AtAMT1;1-eGFP or the empty vector, to generate gap1::WT, gap1::T460A, gap1::AMT1;1-eGFP or Δgap1 strains respectively.
Select transformants on solid YPD medium supplemented with 300 µg/mL hygromycin.
Amplify colonies in liquid YPD and reselect on solid YPD supplemented with 300 µg/mL hygromycin.
Confirm the insertion at the Gap1 locus by PCR and by complementation of functional proteins.
(1) Goldstein, A. L. & McCusker, J. M. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541-1553 (1999).
(2) Marini, A. M., Soussi-Boudekou, S., Vissers, S. & André, B. A family of ammonium transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 4282-4293 (1997).
(3) Gietz, R. D. & Woods, R. A. Tranformation of yeast by the LiAc/SS carrier DNA/PEG method. Methods Enzymol. 350, 87-96 (2002).
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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