Home Introduction Methods Results
Discussion
Spinacia oleracea was chosen to test for Rubisco. The object was to see if there is a difference in Rubisco levels between different brands of spinach, methods of production of spinach, and modes of storage of spinach. We hypothesized that organic spinach would have more Rubisco than conventional spinach. It was believed that the spinach packed in less permeable containers would have more Rubisco than those in more permeable containers. Also, we believed that Fresh Express spinach would have more Rubisco than Dole spinach.
The absorbances obtained in the protein quantification allowed us to determine the concentration of the original sample of spinach. It was determined that FBO spinach had the highest concentration of protein at 1% dilution, followed by DBC, DCS, and DOS, which had the least amount of protein. At 5% dilution, it was FBO had the highest protein concentration, followed by DOS and DCS, which had the same amount of protein, and DBC, which had the least amount of protein. The differences in these concentrations across samples are primarily reflective of the fact that protein was more easy to isolate from different samples. Given that FBO had the highest absorbance reading and DOS had the lowest and assuming minimal experimental error, it is concluded that facilitation of protein isolation is not associated with organic methods of production.
In DNA extraction, the absorbances of the samples were taken to measure DNA content. The absorbencies were taken at 260nm and 280nm. The ratio of A260/A280 was used to determine purity of the DNA. If the ratio is between 1.8 and 2.0, the sample is considered pure. None of our samples fell into that range. The A280 values seemed to be very small, making the ratios very high, suggesting something else was in our sample that absorbed at 260nm.
In Real-Time PCR, the spinach samples were run at two different concentrations (50 ug and 100 ug) on a gel. The melting curves and Ct values for each of the samples were obtained. The Ct values that are high tended to have the least amount of DNA, while the samples with low Ct value were generally from samples with high DNA concentration. These slight trends suggest that DNA isolation was not complete and moreover, some of the impurities were inhibitors of DNA replication. Overall it can be said that the DCS sample had the most DNA, because it had the lowest Ct values in both concentrations.
The PCR samples were run on agarose gel to separate DNA made up in the PCR using primer C. The results showed that all of the samples in lanes 3, 4, 6, and 7, contained rubisco in them, because of the presence of bands at the 200 bp marker for all the samples. Unexpectedly, the data obtained in Real-Time PCR and agarose gel electrophoresis were contradicting each other. The Ct value for FBO would normally indicate that it would have the least amount of DNA present, but in the agarose gel, the FBO strand migrated the least, indicating that it was the heaviest, meaning it had the most DNA. This means that there is more actual starting material for FBO spinach, but more inhibitors as well. It is possible that these observations could be loading error incurred when filling the wells with our samples.
The Western blot was performed to separate the proteins. Two gels were used in this process. The first gel was soaked in Comassi blue to determine the presence of Rubisco, and the second gel was electroblotted onto a membrane to further give information regarding Rubisco. The Comassi blue stain indicated that all four of our samples had Rubisco present with the presence of thick strands. Even more convincing was that the strands were at the same level, telling us that the same protein was expressed in all samples. The electroblotted membrane also showed bands for all four lanes at same spots. This indicated that Rubisco had successfully been located. There were not any visible bands aside from the ones believed to be Rubisco, allowing us to believe that there was no nonspecific binding.
Our hypothesis was not confirmed, because overall, organic produce had less DNA than that of conventional produce. The hypothesis that packaging spinach in less permeable containers would yield higher Rubisco levels also was not confirmed, because there was no observed difference between the absorbances, agarose gel, or western blot results of boxed spinach (FBO & DBC) and bagged spinach (DOS & DCS). The last hypothesis, one that predicted Fresh Express spinach would have more Rubisco than Dole Spinach, was also not confirmed. The Real-Time Ct values show that Fresh Express actually has less DNA than that of Dole. However, these differences are likely to be due to actual differences in DNA levels because it is likely that the amount of protein was similar across condition and therefore all conditions experienced similar amounts of inhibiton.
For future experiments, more samples, but lesser variables, should be tested. For example, a future study could be done solely on the type of packaging of spinach on Rubisco levels. Instead of having only two types of packaging, and four types of plants, one can find several different types of packaging, and see if those have an effect on Rubisco levels of the same species after a fixed incubation time. The same type of spinach used would serve as a control, and would also help determine which type of packaging is best for its longevity. However, in conclusion for this experiment it can be said that all rubisco levels looked the same and thus, we had a mull finding.