1) 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.
2) 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.
3) 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.
4) Amplify this cassette by PCR and clone into a blunted BglII of pDL001.
5) Subclone AtAMT1;1, AtAMT1;1-T460A and AtAMT1;1-eGFP from pDR vectors into the KpnI site of the Gap1 integrative plasmid.
6) Use the yeast strain 31019b (∆mep1 mep2::LEU2 mep3::KanMX2 ura3; see reference 2) to generate DL1 ( Δmep1-3 Δgap1) mutant strain and the versions in which AtAMT1;1 or its mutants are integrated.
7) 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.
8) Select transformants on solid YPD medium supplemented with 300 µg/mL hygromycin.
9) Amplify colonies in liquid YPD and reselect on solid YPD supplemented with 300 µg/mL hygromycin.
10) Confirm the insertion at the Gap1 locus by PCR and by complementation of functional proteins.