Name : Lita Mustika

Student ID : 24020118190145
Recombinant Genetics

DNA Isolation of Yeast Cell

INTRODUCTION
Yeast cells are members of the
Fungus Kingdom. They are single celled
microorganisms (eukaryotic) classified
under phyla Ascomycota (sac fungi) and
Basidiomyota (higher fungi) both of which
fall under the subkingdom Dikarya. Yeast
contains almost the same organelles of a
mature eukaryotic cell. Nucleus, Golgi
apparatus, mitochondria, endoplasmic
reticulum, vacuole, and cytoskeleton are
the most important one. Yeast cell particle
size is typically of 5x10μm. Isolating yeast
at this point is as simple as taking a very
small amount of your culture and rubbing
(streaking) it on to an agar plate. Because
of the stable, non-liquid agar medium,
once streaked, single colonies of microbes
are essentially stranded by themselves. In
this review, I would like to compare
journals, titled “Isolation and
Characterization of Live Yeast Cells from
Ancient Vessels as a Tool in Bio-
Archaelogy” and “Extraction of Genomic
DNA from Yeasts for PCR-Based
Applications”, in order to know and get a
deep understanding about DNA isolation
of yeast cell.
METHOD
Isolating yeast at this point is as simple as
taking a very small amount of your culture
and rubbing (streaking) it on to an agar
plate. Aouizerat et al., (2019) used
conventional and simple method to isolate
the DNA of yeast cells. Conventional
methods for gDNA preparation from yeast
cells utilize either enzymatic degradation,
or beating with glass beads, generally
followed by lysis of cells with detergent
and extraction of gDNA with phenol-
chloroform.
Because of the stable, non-liquid
agar medium, once streaked, single
colonies of microbes are essentially
stranded by themselves. After a few days
or weeks (depending on incubation
temperature and microbe population), they
will multiply and grow large enough to be
seen with the naked eye. At that point, it is
a matter of selecting the colonies you like
and growing them up to larger amounts. It
is impossible to stress enough how
important cleanliness and sanitation are at
this point in the process. If you truly want
a pure strain, you need to ensure you are
not contaminating what you are trying to
isolate with other cultures.
Meanwhile, Lõoke et al., (2011)
developed a quick and low-cost genomic
DNA extraction protocol from yeast cells
for PCR-based applications. On yeast
genomic DNA extraction with LiOAc-
SDS that had done by Lõoke et al., (2011),
the reagents used are 0.2 M Lithium
acetate 1% SDS solution and Ethanol 96-
100% and 70%. First, pick one yeast
colony from the plate or spin down 100-
200 μl of liquid yeast culture (OD600=0.4).
Suspend cells in 100 μl of 200mM LiOAx,
1% SDS solution. Next, incubate for 5
minutes at 70 °C and add 300 μl of 96-100
% ethanol, vortex. Spin down DNA and
cell debris at 15.000 gram for 3 minutes.
Wash pellet with 70% ethanol. Dissolve
pellet in 100 μl of H2O or TE and spin
down cell debris for 15 seconds at 15 000
g. Last, use 1 μl of supernatant for PCR.
RESULT AND DISCUSSION
Aouizerat et al., (2019) found that
several lines of evidence strongly suggest
that the yeast strains they isolated are
indeed descendants of fermenting yeasts in
the ancient vessels. Indeed, testing of
several modern vessels that were filled
with filtered and unfiltered beer and buried
for 3 weeks underground, as well as
further tests of a clay wine vessel that had
not been used for more than 2 years,
revealed that yeast cells can be found in
the clay matrix, after an extended period of
time (Fig. 1A). Next, we tested several
methods of yeast isolation and developed a
pipeline (Fig. 1B) that enabled us to
efficiently isolate viable yeast cells from
these modern clay containers. In contrast,
we could not isolate any live yeast from
the control vessels, which were filled with
filtered beer, nor were yeast cells detected
by electron microscopy
Isolation of yeast from clay vessels. (A)
Yeast strains in clay vessels. Scanning
electron microscope (SEM) pictures of the
inside of a modern clay vessel buried in the
ground for 3 weeks without beer (left
panel) and presoaked with unfiltered beer
(middle panel) prior to burial. On the right
panel is a 2-year out-of-use wine clay
vessel (bottom) that yielded live yeast cells,
observed as colonies and by electron
microscopy (EM [upper panel]). Yeast
cells were only successfully isolated from
the last two vessels. (B) The pipeline of
yeast isolation and characterization from
vessels. Putative fermented beverage-
containing vessels were carefully
dismantled. Small pieces were sent for
scanning electron microscopy (SEM), and
the rest were incubated in growth medium
(YPD) for 72 h at room temperature.
Samples were plated on selective plates
with antibiotics to eliminate bacteria. After
72 h, yeast colonies appeared and were
regrown on new plates. The yeast strains
were taken for various analyses, including
full-genome sequencing and comparison of
growth under fermentation-related
conditions in beer wort. In addition, beer
was brewed according to a standard recipe
using the isolated yeast strains. The
presence of aromatic and flavor compounds
in the beers was analyzed quantitatively,
and their flavor was qualitatively evaluated
by specialized beer tasters.
 Meanwhile, Lõoke et al., (2011) after chloroform extraction, which slows
said that When analyzing large number of down the protocol and makes it
samples these methods are time consuming inconvenient for simultaneous handling of
and relatively expensive. For quick large number of samples. Alternatively,
genotyping cells can be lysed also by gDNA can be prepared in a single tube by
repetitive freezing-thawing cycles in the simple SDS treatment. However, the yield
buffer containing Triton X-100 and SDS, of gDNA by this protocol is relatively low
followed by extraction of gDNA with (Fig. 1B) and the results are poorly
chloroform. Although this method is reproducible (Fig. 1A). In addition, high
considerably faster than conventional number of cells is required for the protocol
gDNA preparation methods, it requires and for subsequent PCR the reaction buffer
transfer of the sample to a new test tube has to be supplemented with Triton X-100.
(A) PCR amplification of S.
cerevisiae gDNA prepared from single yeast
colonies by LiOAc-SDS (method A; lanes
1-8) or by SDS treatment (method B; lanes
7-16). 489 bp and 2383 bp gDNA fragments
from VPS13 locus were amplified with
FirePol® Taq polymerase (Solis BioDyne)
for 30 cycles (95°C; 15 sec – 58°C; 30 sec –
72°C; 150 sec). The reaction buffer was
supplemented with 1% Triton X-100 for
samples 7-16, as recommended by the
authors (4). (B) Total lysate of 1 × 107 cells
prepared by methods A and B was analyzed
on 0.9% TAE-agarose gel. Lanes 1- 4:
dilution series of yeast gDNA concentration
standards prepared by method D; lane 5:
gDNA prepared by method A; lane 6:
gDNA prepared by method B; lanes 7-8:
gDNA prepared by methods A and B were
purified further by RNase A and Proteinase
K treatments, followed by
 phenol:chloroform extraction and ethanol
precipitation of gDNA. Longer exposure of
the gDNA band is shown in the lower
panel. (C-F) Optimization of LiOAc-SDS
lysis protocol conditions. Concentrations of
SDS and LiOAc, lysis time and temperature
were tested (panels C, D, E and F,
respectively). Variable parameters are
indicated below the panels and constant
parameters are shown on top. 489 bp
genomic DNA fragment from VPS13 locus
was amplified by PCR. RT- room
temperature. See figure 2B for DNA size
marker (lanes “M”). PCR products were
analyzed on 0.9 % TAE-agarose gel
electrophoresis, stained with ethidium
bromide and photographed. Sequences of
PCR primers are listed in Supplementary
material.

CONCLUSION that are widely used for analysis of

In summary, Lõoke et al., (2011) have
developed a quick and reliable method for
gDNA extraction from yeasts that is
suitable for PCR amplification of DNA
fragments up to 3500 bp. The protocol can
be carried out in a single test tube within
15 minutes and cells from both liquid and
solid media can be used. The method is
suitable for routine genotyping of yeasts
either by simple detection of PCR
products, or for initial amplification of
genomic DNA for sequencing; procedures
scientific, environmental, industrial and
clinical samples. Meanwhile, Aouizerat et
al., (2019) stated that  isolating, growing,
and studying fermenting microorganisms
from ancient vessels in order to expand
archaeological knowledge of ancient diet
and food-related technologies are feasible.
These results, which allow a more precise
recreation of ancient-like beverages than
ever before, unlock enormous potential for
the study of a broad range of food-related
issues in antiquity. This includes
expanding the knowledge about the technological transfer between them,
ancient diet of diverse societies in many uncovering trade routes and food
periods and locations, the study of the preparation technologies, and even
functions of ancient vessels, facilities, and obtaining insights into the actual somatic
infrastructures, understanding links aspects (aroma and flavors) of ancient
between cultures or identity groups and foods and beverages.

REFERENCES
Aouizerat, T., Gutman, I., Paz, Y., Maeir, A. M., Gadot, Y., Gelman, D., Hazan, R.
(2019). Isolation and Characterization of Live Yeast Cells from Ancient Vessels as a
Tool in Bio-Archaeology. mBio, 10(2).doi:10.1128/mbio.00388-19 
Lõoke, M., Kristjuhan, K., & Kristjuhan, A. (2011). Extraction of genomic DNA from yeasts
for PCR-based applications. BioTechniques, 50(5), 325–328.doi:10.2144/000113672