The insertion Green Monster (iGM) method for expression of multiple exogenous genes in yeast.
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.
Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115.
Vyacheslav M
VM
Labunskyy
Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, La Jolla, California 92037.
Yo
Y
Suzuki
Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, La Jolla, California 92037.
Timothy J
TJ
Hanly
Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, La Jolla, California 92037.
Ayako
A
Murao
Donnelly Centre and Departments of Molecular Genetics and Computer Science, University of Toronto, Toronto, Ontario M5S 3E1, Canada Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, Ontario M5T 3L9, Canada fritz.roth@utoronto.ca vgladyshev@rics.bwh.harvard.edu.
Frederick P
FP
Roth
Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 fritz.roth@utoronto.ca vgladyshev@rics.bwh.harvard.edu.
Vadim N
VN
Gladyshev
D000595:Amino Acid Sequence; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010802:Phylogeny; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D051140:Selenoproteins / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA
D012333:RNA, Messenger; D012269:Ribosomal Proteins; D051140:Selenoproteins; D049452:Green Fluorescent Proteins
Saccharomyces cerevisiae; flow cytometry; green fluorescent protein; multi-gene insertions; synthetic biology