The insertion Green Monster (iGM) method for expression of multiple exogenous genes in yeast

G3 (Bethesda). 2014 Apr 28;4(7):1183-91. doi: 10.1534/g3.114.010868.


Being a simple eukaryotic organism, Saccharomyces cerevisiae provides numerous advantages for expression and functional characterization of proteins from higher eukaryotes, including humans. However, studies of complex exogenous pathways using yeast as a host have been hampered by the lack of tools to engineer strains expressing a large number of genetic components. In addition to inserting multiple genes, it is often desirable to knock out or replace multiple endogenous genes that might interfere with the processes studied. Here, we describe the "insertion Green Monster" (iGM) set of expression vectors that enable precise insertion of many heterologous genes into the yeast genome in a rapid and reproducible manner and permit simultaneous replacement of selected yeast genes. As a proof of principle, we have used the iGM method to replace components of the yeast pathway for methionine sulfoxide reduction with genes encoding the human selenoprotein biosynthesis machinery and generated a single yeast strain carrying 11 exogenous components of the selenoprotein biosynthetic pathway in precisely engineered loci.

Keywords: Saccharomyces cerevisiae; flow cytometry; green fluorescent protein; multi-gene insertions; synthetic biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Phylogeny
  • RNA, Messenger / chemistry
  • RNA, Messenger / metabolism
  • Ribosomal Proteins / chemistry
  • Ribosomal Proteins / genetics
  • Ribosomal Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Selenoproteins / genetics
  • Selenoproteins / metabolism
  • Sequence Analysis, RNA


  • RNA, Messenger
  • Ribosomal Proteins
  • Selenoproteins
  • Green Fluorescent Proteins