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RESULTS: Protein Isolation
Four plants (listed above) were used
in order to test for differences in Rubisco expression between C3
and CAM plants. Sample 1 corresponds to
F.
benjamina,
sample 2 corresponds to
H.
fortunei,
sample 3 corresponds to
K.
daigremontiana,
and sample 4 corresponds to
C.
argentea.
After performing a DC protein assay in which we created a standard
protein curve (by plotting absorbance readings of known protein
concentrations and making a line of best fit), we could then
determine the original protein concentrations of each. We found that
only samples 1, 3 and 4 contained significant levels of protein to
continue on to the next step (Figure 1, Table 1).
Figure 1.
DC Protein Assay Standard Curve.
Table 1.
Absorbance readings and calculated
concentration values. Sample 1 corresponds to
F.
benjamina,
sample 2 corresponds to
H.
fortunei,
sample 3 corresponds to
K.
daigremontiana,
and sample 4 corresponds to
C. argentea.
We calculated the original protein concentration in each sample by
multiplying the "protein in assay" values by a conversion factor of
100, then dividing by the original amount of sample used (for each,
we used 3.33 ul). The "protein in assay" values were found by using
our protein standard curve; since a relationship between absorbance
and concentration was already established using the standard curve,
we could simply go back and plug in an absorbance value to the
standard curve equation to yield the approximate protein
concentration of the sample.
Note that no values can be resolved
for negative absorbance as in sample 2. This is why we did not
continue to use sample 2, which corresponds to the other C3 plant,
H. fortunei.
RESULTS: DNA Isolation
We analyzed each sample afterwards to
determine purity of the DNA. This is important since impure DNA
would result in inaccurate readings of Rubisco expression thus
rendering our conclusions invalid. In order to do this, we took
absorbance readings at 260 nm (corresponding to DNA) and at 280 nm
(corresponding to protein), and looked at the ratio of both
(A260/A280) to determine purity (see Table 2). A ratio between 1.8
and 2 would signify a pure enough sample (higher DNA to protein
ratio). We found that only our samples 1 and 3 were pure enough, but
our sample size was already too small so we decided to go ahead with
the next step in analyzing all 3 samples (1, 3, and 4) through Real
Time PCR (RT-PCR). The DNA concentration of each sample was
calculated by multiplying the absorbance at 260 nm (A260) for each
sample by 50 ug/ml (standard for DNA) and then multiplying by the
dilution factor. Since we diluted each of our samples by combining
100 ul of DNA with 900 ul of H20, our dilution factor for each was
20. Since we ended up with still too high of a concentration for our
samples, we further diluted them by different dilution factors. For
sample 1, we performed a 20 fold dilution; for sample 3, we
performed a 5 fold dilution; for sample 4, we performed a 10 fold
dilution. We did this because too high of a concentration would make
it difficult to mix the correct amount of DNA with water resulting
in a total of 8.4 ul of DNA and water, to then be added to 11.6 ul
of our primer mix per "well" n the RT-PCR step. Please refer to the
Methods
section for more details regarding this procedure.
Table 2.
Absorbance readings for DNA (at 260
nm) and protein (at 280 nm) in each sample. Ratio of DNA to protein
was calculated by dividing A280 into A260 (A260/A280). Sample 1
corresponds to
F. benjamina,
sample 3 corresponds to
K.
daigremontiana
and sample 4 corresponds to
C.
argentea.
RESULTS: Real-Time PCR (RT-PCR)
Real-time PCR was run in order to
quickly and accurately determine which sample contained the highest
amount of DNA coding for the Rubisco gene. This is shown by the
samples' relative C(t) values. Each C(t) value represents the cycle
at which the DNA segment coding for Rubisco would reach a
significant phase of amplication crossing the threshold. Therefore
samples with lower C(t) values are indicative of samples with more
Rubisco gene present, since more DNA with the Rubisco gene would be
amplified exponentially at a faster rate and thus cross threshold
earlier than other samples.
Table 3.
Real Time PCR table*. This data represents our results from the
real-time PCR quantification. Only three of our samples contained
enough DNA to reach the threshold. These samples were a C3
plant-sample 1 (F.
benjamina),
a CAM plant- sample 3 (K.
daigremontiana)
and a nother CAM plant- sample 4 (C.
argentea).
*WELL
NUMBER AND LABELS DECODED:
Note: Some appear twice. This is because each kind
(D1, D3, D4) was run twice.
A12, PC 12.14 ng D3 = (8.40 ul DNA from
K. daigremontiana
--
sample 1 + 0 ul H2O) + 11.6 ul Primer mix
B12, PC 12.14 ng D1 = (6.67 ul DNA from F. benjamina-- sample
1 + 1.73 ul H2O) + 11.6 ul Primer mix
C12, PC 12.14 ng D4 = (5.06 ul DNA from C. argentea-- sample
4 + 3.34 ul H2O) + 11.6 ul Primer mix
D12, PC 12.14 ng D3 = (8.40 ul DNA from
K. daigremontiana
--
sample 1 + 0 ul H2O) + 11.6 ul Primer mix
E12, PC 50 ng D1 = (5.49 ul DNA from F. benjamina-- sample 1
+ 2.91 ul H2O) + 11.6 ul Primer mix
F12, PC 12.14 ng D4 = (5.06 ul DNA from C. argentea-- sample
4 + 3.34 ul H2O) + 11.6 ul Primer mix
G12, PC 12.14 ng D3 = (8.40 ul DNA from
K. daigremontiana
--
sample 1 + 0 ul H2O) + 11.6 ul Primer mix
H11, PC 50 ng D1 = (5.49 ul DNA from F. benjamina-- sample 1
+ 2.91 ul H2O) + 11.6 ul Primer mix
E11, PC 12.14 ng D4 = (5.06 ul DNA from C. argentea-- sample
4 + 3.34 ul H2O) + 11.6 ul Primer mix
F11, PC 12.14 ng D3 = (8.40 ul DNA from
K. daigremontiana
--
sample 1 + 0 ul H2O) + 11.6 ul Primer mix
G11, PC 12.14 ng D1 = (6.67 ul DNA from F. benjamina-- sample
1 + 1.73 ul H2O) + 11.6 ul Primer mix
H11, PC 12.14 ng D4 = (5.06 ul DNA from C. argentea-- sample
4 + 3.34 ul H2O) + 11.6 ul Primer mix
We used
Opticon Monitor software to visualize our RT-PCR data for each
sample. As you can see below, no significant amount of Rubisco-coding
DNA was present in any of the samples except for sample 1, which
corresponds to the C3 plant F. benjamina.
Figure 2. Screenshot of Opticon Monitor software, specifically
for samples 1, 3, and 4. Sample 1 corresponds to the C3 plant F.
benjamina; the two different concentrations of its DNA show up
as the prominent light aqua peak and the prominent royal blue peak.
Sample 3 corresponds to the CAM plant K. daigremontiana, and
sample 4 corresponds to the CAM plant C. argentea. Neither of
these show up prominently in the above.
Figure 3. Zoomed-in screenshot of the above data in Figure 2.
Notice how sample 1's Rubisco-encoding DNA shows up sooner (at an
eariler cycle and therefore lower C(t) value) than any of the other
samples. Sample 1 (F. benjamina, the C3 plant) is indicated
by the aqua and blue lines.
The
only sample which showed a significant peak and accumulation of
product (as seen in the below figure) was F. benjamina, or
the C3 plant.
Figure 2. Opticon Montitor Software visualization of melting
curves of all the samples. Highlighted fluorescent green trace shows
amplication of DNA in Rubisco-coding region for F. benjamina
(sample 1) for one concentration. The downwards sloping green line
represents the melting curve for the primer-dimer and the sharp
green peak in the other jagged trace represents the melting curve
for F. benjamina's DNA. The optimal temperature
to take readings of the cycle (C(t) value) for F. benjamina
would therefore be approximately 80.5 deg. Celsius, between the two
peaks. This is because the second peak represents the melting
temperature of the sample itself, and the first "peak" represents
the melting temperature of the primer-dimers. The primer-dimers
should be melted off, but the sample should not, that is why the
temperature in between both values is significant.
RESULTS: Agarose Gel Electrophoresis (from RT-PCR)
Figure 1.
Real-time PCR generated DNA fragments were taken and then could then
be run on an agarose gel.
Primer C was used. Note the significant presence of bands in lanes 5
and 6, corresponding to the C3 plant F. benjamina.
Lane 1: 1 Kb Plus Molecular Weight Ladder
Lane 2:
C. argentea
(sample 4)
Lane 3:
C. argentea
(sample
4), taken from a different RT-PCR well
Lane 4: Negative control for primer C
Lane 5:
F. benjamina
(sample 1)
Lane 6:
F. benjamina
(sample 1), taken from a different RT-PCR well
Lane 7.
K. daigremontiana
(sample 3)
Lane 8.
K. daigremontiana
(sample 3), taken from a different RT-PCR well
RESULTS: Western Blot
Figure 2.
Polyacrylamide gel depicting proteins stained with Comassie blue.
Lanes 1-4: Other group's data.
Lane 5: Molecular weight marker
Lane 6: Protein isolation from F. benjamina (samp 1)
Lane 7: Protein isolation from F. benjamina (samp 1)
Lane 8: Protein isolation from C. argentea (samp 4)
Lane 9: Protein isolation from C. argentea (samp 4)
Lane 10: (none)
Note:
We did not use sample 3 due to the low concentration of protein in
the sample to begin with, also due to the lack of results from
RT-PCR for that sample. There is a weak signal in all four lanes,
however a slightly stronger signal in lanes 6-7, corresponding to F.
benjamina (the C3 plant).
Figure
3.
Western transfer membrane, post-addition of antibodies (chicken,
rabbit). Lanes match up with Figure 2's lanes. Contrast has been
increased using Adobe Photoshop in order to highlight the fact that
lanes 6-7 (the C3 plant,
F.
benjamina)
depict more signal (analagous to more protein) than lanes 8-9
(corresponding to the CAM plant,
C.
argentea).
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