Protoplasting and Speroplasting research presentation poster for the Jacksonville State University student symposium

1. Novel Methods for Obtaining Spheroplast and
Protoplast in Yeast and Cauliflower
Vanessa Chappell, Levi Brewer, Dr. Roger Sauter
Jacksonville State University, Department of Biology
Grinding:
1. 30g of cauliflower curds were
liquified using sequential
blending techniques in a buffer
at pH 7.5 containing 0.32M
mannitol and 0.019M HEPES in
order to obtain thin sheets of
intact cells.
2. The curds were blended with
short bursts and the
homogenate was strained
through several layers of
cheesecloth to remove the liquid
portion.
3. The final material was
suspended in a pH 5.5
protoplasting buffer containing
0.9M mannitol for protoplasting
Protoplasting:
1. 5g of cauliflower material was added
to 30ml of a pH 5.5 buffer containing
0.9M mannitol buffer and 10%
Viscozyme
2. Sample was incubated at 37*C and
150 rpm for 60 mins.
3. After 60 minutes the sample was
homogenized 5 times and incubated
an additional 30 min to obtain
protoplast
4. After protoplast were obtained 4-6
rounds of 10 second sonication
bursts set at 6 were performed in
order to lyse cells and obtain
organelles for further study.
Cytochrome c, a protein involved in the electron transport chain (ETC), is associated with programmed cell death (PCD) mechanisms, including induction of apoptosis in
vertebrates. Mammalian liver cells were shown to release cytochrome c in response to binding of the mitochondrial membranes by each core histone (H2A, H2B, H3, and H4) and
linker histone H1 [1]. Preliminary evidence has also shown that this same process occurs for histone H3 in Brassica oleracea [2]. We hypothesize that this process is highly
conserved across eukaryotes and provides a method for studying its role in PCD pathways for other eukaryotic organisms. Currently, the research is focused on further describing
histone interactions with mitochondria in B. oleracea and performing preliminary studies to confirm this process occurs in Saccharomyces cerevisiae. Novel methods for obtaining
gram amounts of B. oleracea protoplast and S. cerevisiae spheroplast (cell wall free cells of cauliflower and yeast, respectively) have been developed. Protoplast were most easily
obtained after a series of sequentially blending curds for one-second bursts before cell wall removal with 10% Viscozyme concentration and sonication. Spheroplast were most
easily obtained by harvesting and storing yeast cultures that were grown to an OD600 reading of approximately 0.5. This will allow future cell fractionation and histone purification.
• Cauliflower protoplast were most easily obtained
using sequential blending, 10% Viscozyme™
concentration, and sonication.
• Yeast spheroplast were most easily obtained by
controlling the growth to a maximum OD600 of 0.5
and addition of 2 Lyticase™ units per OD unit.
We hypothesize that histone-mediated cytochrome
c release is highly conserved in plants, fungi, and
animals.
Goals:
• Isolate histones and mitochondria from
cauliflower and yeast cells.
• Incubate mitochondria with purified histones.
• Assay for histone binding and cytochrome c
release by Western blotting.Culturing:
1. Prepared 1 L of culture media in
a 2 L baffled Erlenmeyer flask.
2. Inoculated media with 0.01 g
store bought baker’s yeast.
3. Grew on a shaker/incubator at
25℃ and 100 rpm until a
maximum OD600 of 0.5 was
obtained (See Figure 1).
4. Centrifuged culture at 2600 x g
for 5 minutes.
5. Poured off supernatant and
suspended pellet in yeast
storage buffer before storage at
-20℃.
Spheroplasting:
1. Thawed suspension and
centrifuged at 2000 x g.
2. Poured off supernatant and
suspended pellet in Lyticase™
buffer.
3. Diluted suspension, measured
OD600 of the dilution, and
calculated total OD units of
suspension.
4. Added 60 mM 2-mercaptoethanol
and 1 Lyticase™ (enzyme mixture
formulated to break down fungal
cell walls) unit per OD unit to the
suspension.
5. Placed suspension on a
shaker/incubator at 37℃ and 150
rpm and measured the OD600
every 5 minutes (See Figure 2.1).
6. When OD600 reached 20-30% of
the control, centrifuged
suspension at 2000 x g for 5
minutes (See Figure 2.2).
7. Poured off supernatant and
washed pellet in Lyticase™ buffer
before storage at -20℃.
• Determine cryopreservation techniques for
protoplast and spheroplast.
• Cross-incubate yeast and cauliflower
mitochondria and histones.
• Investigate this process in other model
organisms.
Introduction:
Conclusions: Objectives:Brassica oleracea:
Saccharomyces cerevisiae:
Future Studies:
Acknowledgements:
• Linda Major
• BY 533: Advanced Plant Biology Course
• JSU Department of Biology
References:
1. Cascone, A., C. Bruelle, D. Lindholm, P. Bernardi, and O. Erikson.
"Destabilization of the outer and inner mitochondrial membranes by core and
linker histones." PLoS One7, no. E35357 (2012).
2. Major, Linda A. Histone binding to mitochondria in plants. Master's thesis,
2014.
Figure 1: Yeast Growth Curve: Yeast growth was monitored by
spectrophotometric analysis every hour using a wavelength of 600
nm. This is an example of a full growth curve, including exponential,
lag, and death phases for yeast. The optimum OD600 for future steps
was obtained around 0.5, and happened at approximately 18 hrs.
Figure 2.1: Spheroplast OD600 Curve: Cell wall removal was
monitored by spectrophotometric analysis every 10 minutes using a
wavelength of 600 nm. From a starting OD600 of 0.5, a gradual decline
to approximately 0.12 confirms that the cell wall has been removed
before enzymatic activity slows after 30 minutes.
Figure 2.2: Spheroplast Percent Control Curve: With reference to a control
solution with no added Lyticase, the spheroplasted cells were divided by
the control to generate a curve resembling Figure 2.1. Before cell wall
removal, the culture suspension was 100% the control. When enzyme
activity slowed at 30 minutes, the suspension reached 26% of the control.
1 4
2
3
5
Fig 1: Thin layers of starting material. Stained with Acetic orcein. The
rectangular or cube shaped cells are easily visualized at 400x
Fig 2: Cauliflower cell sample after 20 minutes incubation. The cells are
starting to separate. Some are starting to loose their rigid shape.
Fig 3: Cauliflower sample after 40 minutes incubation. Most cells have
space around all sides. Most are taking on a spherical appearance.
Fig 4: Cauliflower sample after 60 minute incubation and 5x
homogenation. Cells have definite separation and are uniformly sperical
Fig 5: Cauliflower sample after 90 minutes incubation and 4-7 rounds
sonication. Cells have been lysed open (though a few intake cells remain
visible) These organelles are ready for further testing.