Article: “ The Insertion Green Monster (iGM) Method for Expression of

Multiple Exogenous Genes in Yeast

1. In this article, how are the multiple exogenous genes are detected? What methods are
used? Explain the mechanisms of the method indicated in the journal.

According to the article, multiple exogenous genes are detected by using a complex
exogenous pathway with yeast as a host. In addition, it is frequently desired to knock off or
replace many endogenous genes that may interfere with the processes being researched while
adding multiple genes. The lack of tools to generate strains expressing a high number of
genetic components has impeded research of complex exogenous processes utilizing yeast as
a host. It's common to want to knock out or replace multiple endogenous genes that might
interfere with the processes being examined, in addition to adding many genes.

It was also described in the article that the utilization of the "insertion Green Monster" (iGM)
set of expression vectors was the method used, which allows for the exact insertion of
numerous heterologous genes into the yeast genome in a quick and repeatable manner, as
well as the simultaneous replacement of selected gene of yeast. However, they used and
adapted a previously published technology (the "Green Monster" method) that allows the
assembly of several changed loci into a single strain, with each strain bearing a quantitatively
selectable green fluorescence protein (GFP) flag.

The Green Monster approach has been used to remove many genes from a single strain
before, but not to create multiple loci, each of which carries an external gene. We use this
technology to manufacture gene insertions, which expands the method's adaptability.
Because the introduced genes are placed at unlinked loci in S, the new insertion Green
Monster (iGM) technique is particularly helpful for analysis of numerous genes of a
complicated exogenous pathway in a sequential fashion. cerevisiae. Accordingly, they have
employed the iGM approach to combine 11 foreign genes encoding components of the
metazoan Sec biosynthesis and insertion pathways into a single strain in a proof-of-concept
experiment.

2. How was the universal GFP cassette of gene insertion formed?

A universal GFP cassette of gene insertion was formed when the plasmid pYOGM081
contains the gene insertion module. It contains sequences flanking a URA3 transformation
marker, a GFP reporter gene, and a set of sequences allowing the overexpression of a gene of
interest that are homologous to the ends of the KanMX4 marker for gene targeting. On either
side of the gene insertion module, eight restriction sites (NheI, SacII, SnaBI, SexAI, SacI,
BglII, PacI, and AscI) are present, allowing one or more of the corresponding restriction
enzymes to create free ends that enhance homologous recombination without destroying the
insertion module for many genes. The transcriptional terminator from the ADH gene is
followed by the duplicated tet operator (tetO2), the GFP coding sequence GFP(S65T), and
the CYC1 gene terminator, in that order (Suzuki et al. 2011). The overexpression sequences
were taken from the plasmid pAG416GAL-ccdB-HA (Alberti et al. 2007). The inducible
promoter GAL1-10pr, a Gateway-compatible cloning site (Life Technologies), a HA-tag
sequence, and a stop codon are all included. In pYOGM081, the CYC1 terminator from
pAG416GAL-ccdB-HA is substituted by the TEF terminator. 

3. How is the article related to our lecture?

The article is related to our lesson about the expression of genetic materials since the
process being used in the article relates to transcription and then to translation. In the
generation of universal GFP cassettes for gene expression, expression of genetic material
is being observed where there is a collection of sequences that allow a gene of interest to
be overexpressed. Transcription was actually mentioned in the last part of their method,
which is the mRNA sequencing, where the protein expression was induced by adding 2%
galactose. Then lastly, translation was done in which RNA-seq reads were aligned to
the S. cerevisiae genome from the Saccharomyces Genome Database. 

4. Give a flow diagram of the methods used in the paper.

The figure above shows the iGM insertion module is being tested. (A) The human
RPL30 gene was cloned under the control of the GAL1-10 promoter and coupled to
an inducible GFP and the URA3 marker, replacing the yeast YFR057W ORF. The
positions of the genotyping primers are indicated by arrows. (B) Western blotting
with HA-tag–specific antibodies revealed expression of the human RPL30 gene in a
strain carrying the RPL30 insertion module. Galactose was used to promote the
protein's expression. (C) LC-MS/MS identification of peptide sequences in a yeast
strain bearing the RPL30 insertion module. RPL30 protein was immunoprecipitated
with a HA-tag antibody, separated on SDS-PAGE, and trypsin digested in-gel. The
mass spectrometry-detected peptides are displayed in green. (D) Mouse MsrA protein
expression in the 3 Msr strain lacking all three Msr enzymes. The 3 Msr strain
received an insertion module containing the mouse MsrA gene under the control of
the GAL1-10 promoter, and production of the protein was confirmed by Western
blotting using HA antibodies. (E) On media containing a supply of Met-SO,
expression of the mouse MsrA recovered the development of a 3 Msr strain lacking
all three yeast Msrs. Yeast strains were tested for growth on Met-free SC medium
supplemented with 20 mg/liter Met (left) or Met-SO media supplemented with 20
mg/liter Met (right) (right).

Cells were cultivated on SC liquid medium until their OD600 reached 0.6, then
harvested, washed, diluted in water to an OD600 of 0.1, and serially spotted on agar
SC plates containing galactose and respective Met or Met-SO sources. The plates
were incubated at 30° for 48 hours after plating and pictures were obtained. (F)
Alternative promoter expression of the mouse MsrA protein utilizing iGM vectors.
Insertion modules containing the mouse MsrA gene under the control of the ADH1,
TEF, and CUP1 promoters, as well as a version of the plasmid containing the GAL1-
10 promoter but without the HA-tag, were integrated into the YER042W locus, and
protein expression was detected by Western blotting with either HA-tag or MsrA-
specific antibodies. Logarithmically growing cultures were stimulated to generate the
protein by adding 100 M CuSO4 or 2% galactose for 4 hours where indicated.

https://academic.oup.com/g3journal/article/4/7/1183/6025895
https://www.takarabio.com/learning-centers/cloning/applications-and-technical-
notes/simplified-insertion-of-a-gfp-encoding-cassette-into-a-100-kb-plasmid