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Results

            In order to contribute to the scientific understanding of Rubisco expression in plants, two varieties of plants were obtained from two different times of day.  About two grams of the shoots of an ice plant and oak leaves were obtained during the day (12:00 pm) and at night (6:00 am) to be used in the study of Rubisco.  In order to first start quantifying Rubisco expression, the protein had to be isolated and analyzed.  A DC protein assay (BioRad) was used to determine how much protein was to be used later in Western Blotting.  In the second step of this assay, Folin-Coicalteu was reduced to a blue colored compound being detectable in a spectrophotometer at A750.  Plant tissues were ground up and centrifuged to separate the proteins to be used in the DC protein assay, and to be stored for later use in a Western Blot.  Protein standards were used to create a standard curve of protein concentration of known samples at A750 to be used to convert absorbance of the samples into protein concentrations.  Three different dilutions of each sample were prepared to ensure that at least one fell on the standard curve.  The dilution factors were as follows: 70µL of buffer and 30µL of protein, 95µL of buffer and 5µL of protein, and 99µL of buffer and 1µL of protein. The protein standards and the samples were put in a spectrophotometer and the A750 were recorded as follows.

Table 1:  Protein standards in mg/ml and the A750 recorded from a spectrophotometer. 

Table 2:  The A750 of each dilution of each sample. 

The following graph was created of the protein standards vs. A750.

Figure 1:  Graph of data from Table 1 of the protein standards vs. A750.  Shows trend line and R² value. 

In order to find the total concentration of protein in our samples, only the A750 of the dilutions that fell within the protein standard curve were plugged into the protein standard equation, y = 0.296x – 0.0672, as y and solved for x.  These numbers were then multiplied by their dilution factor to obtain the total protein concentration.  The following numbers were obtained. 

Table 3:  Total protein concentrations of dilutions that fell within the protein standard curve obtained from the protein standard curve equation.  The average for the final total protein concentration of each sample is shown. 

The numbers used for the average total protein concentration were selected because they fell within the standard protein curve as seen by comparing the ABS of each sample to the ABS range of the protein standards.  The total protein concentrations would later be used to figure out how many µL (<30µL) could be loaded in the Western Blot gels to load 30µg of protein.

In the next part of this experiment, a Western Blot procedure was performed.  Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS PAGE) was used to separate the proteins based on size. The proteins were transferred onto a membrane using electrophoresis.  The protein concentrations determined were used to determine how many µL of our protein samples we needed to load 30µg of protein (not exceeding 30µg).  From the average total protein concentrations it was impossible to load 30µg of our protein samples without exceeding 30µL.  The following amounts were determined to be loaded. 

Table 4:  Amount of each sample determined to be run in the Western Blot. 

NI being significantly less concentrated could load a maximum of 0.22µg of protein without exceeding 30µL.  For the other three samples, it was determined how much protein would be in 30µL of NO (the least concentrated of the three), 0.71µg, and then it was figured out how many µL of the DI and DO samples would also contain 0.71µg of protein in order to make a comparison.  Two gels were set up and run at 100 volts until the bands were about two-thirds finished.  One of the gels was submerged in Coomassie blue stain and then destained in water.  The following day a picture of the gel was taken using BioRAd imager. 

Figure 2:  Coomassie blue stained gel showing our samples in the first four lanes and the marker in the sixth lane. 

As seen in Figure 2, the Coomassie blue stained gel did not result in any clear bands.  The second gel was soaked in 1 X Western Transfer Buffer while the membrane was prepared.  The gel was then set up in the Gel Sandwich and ran overnight.  The membrane was then washed with TTBS and placed in a refrigerator with blocking solution to be used in antibody detection and Western Blot analysis.

            For the antibody detection and Western Blot analysis, two different antibodies were used to detect our protein.  The primary antibody, used first, was made in a chicken against a fairly conserved region of the large subunit of Rubisco protein.  The secondary antibody, used second, was produced in a goat that binds to all chicken antibodies.  The second antibody produced the color in order to detect our product.  After washing the membrane several times in TTBS it was suspended in blocking solution containing the primary antibody.  It was then washed several more times in TTBS to get rid of any primary antibody that was not properly bound to the membrane.  The membrane was then suspended in diluted secondary antibody.  It was again washed several times in TTBS.  The membrane was then stained and photographed.  The picture of the membrane did not show any product on our membrane.        

            The next part of the experiment was genomic DNA purification.  The purpose was to isolate genomic DNA from our samples to be used for PCR.  This was achieved by first breaking the cell walls and lysing the cells in order to extract the DNA.  Chloroform/octanol was used to extract the DNA, which acts to bind complexed proteins and polysaccharides making them dense so that they could be centrifuged out.  Isopropanol and salt were then used to precipitate the DNA which causes the DNA strands to condense and become visible.  The DNA could then be separated and was washed and centrifuged several times for purification and finally was resuspended in TE buffer to rehydrate the DNA.  The following week, the DNA concentrations were determined by measuring the absorbance at A260, and the total DNA concentration was calculated using the following formula:  A260 * Eb * dilution factor = total DNA concentration (Eb = 50µg/ml, constant; dilution factor, 20µL of DNA in 980µL of DI H₂O).  The following data was obtained.

Table 5:  A260 of 20µL of DNA in 980µL of deionized H₂O (DI H₂O) of each sample and the total DNA concentrations calculated by A260 * Eb * dilution factor = total DNA concentration (Eb = 50µg/ml, constant; dilution factor, 20µL of DNA in 980µL of DI H₂O). 

The absorbance was also taken at A280 because at that wavelength proteins and nucleic acids absorb light.  Therefore, the ratio of A260/A280 gives the purity of DNA (1.8-2 is good).  The following data was obtained.

Table 6:  A280 of 20µL of DNA in 980µL of DI H₂O of each sample and the purity level calculated by the ratio of A260/A280.

            In the next part of the experiment real time PCR was performed on the DNA to quantify the amount of chloroplast DNA which contains the gene for Rubisco large subunit.  Several PCR reactions were set up using sybr green dye to detect the PCR product, which fits between the bases of double stranded DNA, permitting the detection of how much product is formed.  We set up 14 reactions for which we chose two different primers, and calculated how much DNA to use in each.  We chose to use two of the universal primers that should recognize the Rubisco large subunit, rbc12f/RBCL-fonfana which should give a fragment that is about 500 basepairs (bp), and rbcl2F/RBCL-Savolainen which should give a fragment that is about 200 bp.  It was recommended to use 50-150ng of DNA; therefore, we chose to use 100ng for each sample and an additional reaction of the DO and NO samples with only 50ng.  Only DO and NO were ran with only 50 ng of DNA due to space restrictions because of their low purities.  The master mixes were set up for each primer and the reactions were set up, one for each primer.  Real time PCR was then run for each reaction which produced the following data.

Table 7:  Data from the PCR reactions using primer rbc12f/RBCL-fonfana.  The amount of DNA and which sample used in each reaction is indicated.  The C_T values are also given.

Figure 3:  Graph of the data in Table 7 showing the cycle number that each sample peaked at. The sample colors correspond to the colors in Table 7.

 Figure 4:  Graph of the melting curve for the PCR reactions using the primer rbc12f/RBCL-fonfana.  The data from this graph was used to determine what temperature to take the data in Table 7 and Figure 3 from.

The data in Table 7, and Figures 3 and 4 was obtained from the PCR reactions using the primer set rbc12f/RBCL-fonfana.  Figure 4 indicates what temperature the cycle numbers should be read at, around 82º Celsius.  Table 7 and Figure 3 show each samples’ C_T value indicating which sample contained the most Rubisco.  From these PCR reactions, the order of the samples (with 100ng of DNA) containing the most to the least Rubisco was as follows, NI (CT 9.177), DO (CT 10.133), NO (CT 13.346), and DI (CT 13.454).  The CT values for the DO and NO samples with 50ng of DNA had a similar trend to the DO and NO samples with 100ng of DNA, they just had a lower Rubisco expression with CT values of 10.713 for DO and 14.701 for NO.

Table 8:  Data from the PCR reactions using primer rbcl2F/RBCL-Savolainen.  The amount of DNA and which sample used in each reaction is indicated.  The C_T values are also given.

Figure 5:  Graph of the data in Table 8 showing the cycle number each  sample peaked at.  The sample colors corresponding to the colors in Table 8.

Figure 6:  Graph of the melting curve for the PCR reactions using the primer rbcl2F/RBCL-Savolainen.  The data from this graph was used to determine what temperature to take the data in Table 8 and Figure 5 from.

The data in Table 8, Figure 6, and Figure 6 were obtained from the PCR reactions run using the primer set rbcl2F/RBCL-Savolainen.  Figure 6 indicates what temperature the cycle numbers should be read at, which, for this set of reactions is unclear.  Table 8 and Figure 5 show each samples’ C_T value indicating which sample contained the most Rubisco.  From these PCR reactions, the order of the samples (with 100ng of DNA) containing the most to the least Rubisco was as follows: NO (C_T 18.013), NI (C_T 14.396), DI (C_T 14.390), and DO (C_T 16.813).  The C_T values for the DO and NO samples containing 50ng of DNA were 17.198 and 21.326 respectably.

            The DNA fragments generated by PCR were then separated using agarose gel electrophoresis to estimate the size of the fragments.  The samples were prepared by adding tracking dye and a molecular size standard was also prepared.  The gels were run and then stained with ethidium bromide, and then photographed producing the following picture.

Figure 7:  Photograph of the agarose gel electrophoresis of the DNA fragments from PCR with lane designations as follows: lane 1, NI, 100ng, primer rbcl2F/RBCL-Savolainen; lane 2, NO, 100ng, primer rbcl2F/RBCL-Savolainen; lane 3, DO, 100ng, primer rbcl2F/RBCL-Savolainen; lane 4, Molecular Marker; lane 5, Blank, primer rbc12f/RBCL-fonfana; lane 6, NI, 100ng, primer rbc12f/RBCL-fonfana; lane 7, NO, 100ng, primer rbc12f/RBCL-fonfana; lane 8, DO, 100ng, primer rbc12f/RBCL-fonfana.  Band sizes are indicated. 

The samples in the first three lanes contain the primer set rbcl2F/RBCL-Savolainen should have produced 200bp fragments and the samples in the last three lanes containing the primer set rbc12f/RBCL-fonfana should have produced 500bp fragments.  The first three lanes all appear to have bands around 200bp; the last three lanes all appear to have bands around 500bp.

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