Discussion
Our overall goal for this project was to find the difference, if any, in the expression of the Rubisco protein between deciduous plants and evergreens. The deciduous plants chosen were Maple and Oak trees. American Holly was the choice to represent evergreens. Our hypothesis was that the amount of Rubisco in the American Holly would be greater on account of the Fall season and decreasing rates of metabolism in deciduous trees as the leaves begin to die and fall from the trees. The leaves of these plants were used to extract molecular data to test our hypothesis in various steps of this project.
After extraction of the DNA, the samples which were thought to be purified had absorbencies taken at 260nm and 280nm of wavelength to test for quantity and purity. As seen in Table 3 the Holly seemed to have the most pure sample with a 260nm/280nm ratio of 1.8. Normally samples should be anywhere between 2.0 and 1.8 for good quality. However Maple and Oak both fell quite short of this with ratios at 1.4 and 1.1 respectively. By multiplying each samples 260nm reading by 2,500 (50 for dilution and 50 per 1 OD) an estimation of each samples DNA quantity/concentration is obtained. Maple and Oak’s estimations were 0.775 ug/uL and 1.29 ug/uL respectively. However, since neither of these samples had a good 260nm/280nm ratio it is very likely the actual concentrations of DNA are much lower than this. Holly’s estimated DNA concentration came to 1.37 ug/uL which should be accurate with a 1.8 260nm/280nm ratio.
The second result received from this project was Real-Time PCR analysis from DNA extraction. The absorbencies taken for each sample at 260nm of wavelength were used to quantify the DNA needed for the PCR analysis (Table 3). This DNA was then used in Real-Time PCR with primers made specifically to amplify the Rubisco gene from the plant’s genome. It was very difficult to draw any type of conclusion from the results of this procedure (Figures 2 and 3). As the figures show, only the 25ng Holly sample and the primer (negative control) rose above the computed C(t) value. This means only these two samples had a significant amount of DNA cloned. Having the Holly show positive amplification was a great sign, however with our so called "negative" control showing a higher amplified concentration than the 25ng Holly sample it is not clear what exactly can be determined from this. It could be assumed that for a trial to have a positive conclusion it would need to rise above the negative control and not only the computed C(t) threshold value, otherwise whatever went wrong with the negative control could have just as easily gone wrong with the 25ng Holly sample. This seems to indicate our negative control had more than likely been contaminated somehow. If this is the case and faulty lab work was a likely problem in the PCR step of this project, it may also have been the problem which caused all samples besides the 25ng Holly to not be amplified. The Holly sample only had one peak on its melting curve graph (Figure 5) indicating no sign of primer dimers or multiple DNA samples being amplified. This once again is confusing data as there was the consideration of contamination to this sample. However if this sample had been contaminated with some outside source (such as someone's spit) it would have likely had several peaks on this temperature vs Fluorescence graph.
DNA was taken from these Real Time PCR reactions and run on an agarose gel as seen in Figure 6. The two most prominent bands on this gel, besides the marker in lane 3, were in lanes 2 and 4 which is the Holly DNA and the DNA retrieved from the primer sample respectively. From the results of PCR it was expected to get a visible band of DNA in the Holly lane and a band almost twice as bright in the primer lane. Finding a band in the Holly lane practically as bright, if not just as bright, as the one found in the primer lane leads to questions as to why the PCR analysis showed much smaller quantities of Holly DNA being amplified as compared to in the primer well. If the PCR analysis shows DNA in the primer sample and a much smaller amount of DNA from the Holly sample why do the bands seem practically identical? This cause of this result was unable to be determined. In the 5th lane there was a smear of DNA from the Maple sample. This smear could be some kind of contamination or the sample could have been degraded. There was no band in the 6th lane where the Oak sample was loaded which seemed to confirm the PCR analysis results which showed no DNA amplification.
The Western Blot results seemed promising by the looks of the image taken of the SDS PAGE (Figure 7). The Holly lane had a very strong band in between the third and fourth band of the Kaleidoscope protein standard. However this band was not linear but had a slight curve to it making it difficult to label exactly how many Kilodalton's (Kd) it is. The third and fourth bands of the standard, from the top, are around 57 kd and 30 kd respectively. This means the band from the Holly sample is in the range Rubisco would be found, 55 kd. The Maple also seemed to have a slight band in that same region showing a possibility of also having isolated the Rubisco protein.
The Antibody Detection however did not work out as expected. Separate antibodies were used in this group from those in the other groups on a volunteer basis. A primary antibody of anti-Rubisco from a rabbit and secondary antibody of donkey anti-rabbit were used to try and possibly create a new protocol for use in the labs in upcoming semesters. As seen in Figure 8, the image taken of the membrane shows absolutely no luminescence. At first it was thought that the new antibodies were ineffective but after further discussion with the lab instructor it was found out there were two separate detection reagents used for different groups. One of the detection reagents was only a couple weeks old where the other reagent was over a year old. Only one group used the new reagent and the rest of the groups, including this one, used the old reagent. All groups that used the old reagent had completely blank pictures for their membranes. The one group which used the newer detection reagent received luminescent bands on their image. With this information it seems the older batch of detection reagent was no longer good and therefore we are unable to determine which sample actually had Rubisco protein or how much. However technically this cannot be proven and making the result that no Rubisco was isolated from any sample. This leaves the overall answer to this project, of whether deciduous plants or evergreen plants contain more Rubisco, to be inconclusive. To determine a true answer the protein extraction and purification would have to be performed again and have new Western Blots with antibody detections run.