Discussion
The overall goal of this experiment was to test for varying levels of Rubisco expression in Brassica oleracea and Petroselinum crispum. In order to test for Rubisco in the four plant samples collected, protein and DNA was extracted from the plants and run in various tests to quantify it. We hypothesized that the organic plants would have more Rubisco than their non-organic counterparts because of a higher chloroplast content and lack of chemical stress of traditional farming methods.
The first step was to determine if there was evidence of protein in the samples. A consistent concentration for the four samples was used to better quantify and make our results more accurate that it was actually Rubisco that we were extracting. SDS-PAGE was done to answer this question. As seen in Figure 2, the gel was a mess. The four samples were in the four right lanes and it just looks like a blob or smear of protein and not the band pattern that would have been preferred. The samples in their respective lanes from the ladder (which was in lane 5 coming from the left hand side of the gel) outwards are as follows: Lane 1 had non-organic collared greens, Lane 2 had organic collared greens, Lane 3 had non-organic parsley and Lane 4 had organic parsley. There was clearly something wrong with the way the gel was cast or had some other problem that was undetectable because different pipette angles and techniques were all tried but the samples in the wells would not stay put and therefore, the gel lanes almost look slanted for the entire thing. The samples just kept drifting over into different wells for no apparent reason. It is hard to detect on the webpage, but if the hard copy of the picture is closely examined, there appears to be a band present in at least three out of the four samples at the 55kb spot, if not all of them. The marker seems to have turned out fine. The only data that could be taken away from this gel is that there has to be protein present because there is a smattering of it all over our four lanes. Since 30μg of protein was loaded into each well of the polyacrylamide gel, based on determining the total protein concentration by DC Assay and a standard curve (Figure 1, Table 1) the concentrations are normalized and therefore the intensity of the RUBISCO band indicates expression levels in each sample. On this gel, it is hard to look at band intensity because it is so messed up that we decided to focus on lane intensity. Lane 2, containing the organic collared greens appeared to have had the most protein due to the very deep intensity of the protein smear. Lane 4, the organic parsley also had a deep smear suggesting that from this first test, the organic plants have a larger concentration of Rubisco than their non-orgainc counterparts. The results are lackluster at best due to the bad gel and the few inferences mentioned previously are the best that can be taken away from this SDS-PAGE gel.
A Western Blot is used to confirm that a specific protein is present, so this was the next test to determine if the protein isolated actually contained Rubisco. The Western Blot membrane was hybridized with two antibodies that specifically detect Rubisco. For the Western Blot, the same amount of protein was loaded into the wells. Again, this gel (Figure 3) appears to be a mess, at least for our four lanes. This could possibly be attributed to the proteins being kept frozen in liquid nitrogen for a week longer than they usually are. Western Blots are the most accurate when the proteins are very fresh and have not had much time to degrade. The lanes are in the exact same order as the SDS-PAGE gel; however our samples are in the first four left lanes for this gel. Interpreting this gel is very difficult because it appears that we just got a very big cluster of protein across the four lanes. A very big leap of faith says that organic parsley had the most Rubsico protein due to the size of the band on the gel, so going by this it also supports our hypothesis. It is hard to say that the data is inconclusive because there is definitely protein present, however, due to it not really being a band it is hard to say if it is definitely Rubisco protein and that they are quality results. It is very possible that human error in could have been introduced into the protocol attributing to the bad quality of protein on the gel. Another possible source of error, could be an improperly formed gel. When loading the gel various research has trouble with the wells, indicating there was debris, this could have prevented samples for loading correctly and subsequently altered how they ran on the gel. Additionally a leak in the gel apparatus could have cause the gel to run improperly.
The DNA isolation was preformed to clarify that data obtained from the Protein Isolation. We were only able to get DNA isolates from three of the four samples (Non-organic Collard Greens, Organic Parsley, Non-Organic Parsley) due to an experimental error in the isolation procedure. The rt-PCR experiment gave only one result per three triplicates, again likely due to experiment error in the set up of the reactions. However that data that was obtained followed the characteristic log growth pattern and exhibited the expected melting curve. Judging from the melting curve graph (figure 4c), the plate read chosen to analyze the results was 80*C, because this point removed all of primer dimer/ non-target DNA products melting to the left, and left only the large spike from the target RBCL product. It was confirmed by gel electrophoresis (figure 5) that RBCL was actually the DNA amplified in the rt-PCR experiment. The data from the rt-PCR showed distinct CT values for all three samples (table 2). It was found that non-organic collard greens had more RBCL than did organic or non-organic parsley, NOCG had CT value of 16.477 while the OP and NOP had CT values of 22.567 and 23.589 respectivly. This indicates that RBCL expression varies widely by species and to compare the effects of farming method only one species should be compared at a time. The parsley data did confirm the hypothesis that organic farming yielded higher RBCL levels, but the CT values differ by roughly 1. This difference is not conclusive, especially because only one sample was amplified from each, so there are not multiple trials for comparison. However this is a start to providing information on organic farming, and a future rt-PCR experiment could be run using more samples (n=10) to determine if this pattern in RBCL expression is universal.
It should be noted that all groups had difficulties with the DNA isolation procedure. Expermental error is not a reliable explanation, as all groups had the same problem. The cholorform extraction phase was reversed in our experiment and resulted in less DNA being harvest. The most likely explanation of this reversal is the reagents. Perhaps a reagent was mixed incorrectly or was mislabeled, this could explain the variations in density observed.
Based on all of the information, we can not fully support or reject our hypothesis. Instead we propose increased testing using more samples. Other measurements of transcript levels could also be utilized, such as Northern Blot or Microarray to futher support the tests used in this research. In conclusion we propose a revised hypothesis, that farming method does not have a significant effect on Rubisco expression.