Purity was consistent among species, albeit a low purity for the Nirvana specimens  Table 1 .  Modification to procedure of DNA isolation would probably remedy this to produce higher DNA/protein ratios.  A high protein concentration accounts for the low purity, absorbing light at 280 nm and lowering the ratio DNA/protein.  Using the absorbance of our samples at 260 nm, DNA concentration was calculated.  This was an essential step to interpreting the results as confined to just DNA.

     Chloroplast content was determined by using Real Time PCR to compare Rubisco gene copies. Real Time PCR was used to amplify the Rubisco gene.  Impatiens grown in the shade had the lowest starting concentration of the Rubisco gene (Figure 1).  Impatiens grown in the sun has the lowest Ct of 2.8, an indication that Rubisco gene was present in highest amounts (starting concentration) out of all our samples (Figure 1). The Nirvana species grown in sun has a lower starting amount of the RUBISCO gene than Nirvana grown in shade (Figure 1). Real Time data does support our hypothesis in terms of the Impatiens species, however, the Nirvanas does not coincide with our hypothesis because it indicates that there were more chloroplasts in leaf tissues grown in the shade than in the sun.  The blank control with primers only amplified DNA due to its Ct value of 24.4, pointing to a mild degree of contamination.

      Melting temperatures for our DNA samples (Tm) (Figure 2) indicated that the suitable temperature of annealing for PCR is ~ 81.5 degrees. The Real Time PCR reaction, with standards (Figure 3), allowed for the semi-quantization of our sample.  Our original amounts of the Rubisco gene are as follows:  Impatiens sun (not able to be determined), Impatiens shade ~ 256 pg, ~ 400 pg Nirvana Sun, ~512 pg Nirvana Sun.  These amounts are very generalized since some standards were discounted due to ineffective PCR reaction.

     Utilizing a SDS page and Western Blot technique we could compare the level of gene expression between the four types of  plants and their designated growth conditions. It was hypothesized that Rubisco gene expression would be lower in plants with higher Chloroplast content. Although the following does not support or refute our hypothesis, Rubisco expression can be compared in the Nirvana species.

    Group generated standards, allowed us to generate a standard curve (Figure 5 B) and equation from which sample concentration could be calculated.  Sample 1.1 is Impatiens shade at 1:2 dilution, 2.2 is Impatiens sun at 1:20 dilution, 3.3 is Nirvana shade at 1:200, 4.2 is Nirvana sun at 1:20.  Each sample dilution was chosen based on how well its absorbance fit on the graph in Fig 2.  Knowing sample concentration allowed for consistent amounts of protein to be loaded into the Western Blot, which allowed us to make accurate conclusions about Rubisco in our hypothesis.

      The combined figures SDS-Page electrophoresis gel Figure 6 A and Western Blot Figure 6 B show that Rubisco is present in our samples.  The marker for Western Blot ran faintly, however for confirmation by comparing the marker from the Comassie Blue the Western Blot data can still be useful.  Assuming the lower bands are of the correct weight (Comassie Blue electrophoresis gel), the appearance of Rubisco bands can be estimated.  The positive control for Rubsico ran fine on the Western Blot, also helping us to estimate bands.  Higher amounts of Rubisco are visible in Nirvana, more specifically Nirvana grown in sun.  Nirvana grown in shade has a slightly fainter band.  This does not support our hypothesis that plants grown in sun will produce lower levels of Rubisco.  It is however impossible by these results to determine how much transcriptional activity is taking place per chloroplast. Also it is uncertain as to whether any further hypothesis can be made from the limited amount of data we have for protein expression among all samples.  Bands for Impatiens did not show, this may be because the bands were not amplified enough to be detected by the camera, or problems in protein isolating may have occurred due to leaf consistency (thick leaves and very slimy in solution form).

      Many experiments can be done to expand on our original hypothesis.  Another venture would be to test the same hypothesis on C3,C4, and CAM plants and compare the results.  The C3 plants method of carbon fixation is best suited to areas with high CO2 concentrations, C4 and CAM  plants are able to fix carbon in the presence of lower concentrations of CO2.  This would give insight on how plants use adaptations in RBCL and chloroplasts levels to fix carbon in various environments. 

      Similarly, this type of experiment need not be limited to terrestrial plants.  Aquatic species can be tested for RBCL and chloroplast variation due to dissolved CO2 in water.  In addition, methods for an experiment using aquatic plants may allow for more control over dissolved gas levels due to aerators and water quality testing equipment available for aquariums. 

1. PCR Results

2. Western Blot

3. Future Ideas

Title Page

Introduction

Methods

Results

Literature Cited