rewrite the result and discussion

Results

The two plasmids inserted into bacterial cells each had their own functions. The guide plasmid ensures the template plasmid get inserted in the right place. CRISPR CAS9 works by cutting the region of DNA that new information needs to be added in (Figure 1). The PCR step was verified using the gel electrophoresis (Figure 2). The first column is the ladder which is like the standard for measuring the quantity of plasmid taken by our bacteria.

The staining part of this experiment was a verification to ensure the transfection process was successful, by comparing the treated and untreated HeLa cells. As seen in the image both treated and untreated cells have a nucleus, which can be seen when stained with the DAPI stain (Figure 3). Also both type of cells had the actin protein which was seen when the cells where stained with phalloidin. The treated HeLa cells were the only ones able to show red fluorescent proteins under the microscope (Figure 3).

BCA assay standard curve provided a linear model in order to calculate the protein concentration from the absorption. In order to calculate the concentration, the absorbance number is placed in the equation. The absorbance input rendered a concentration of 540.0926 ug/mL (Figure 4). Another way to find the concentration is to find the point where the absorbance on the x-axis and the concentration on the y-axis meet on the line (Figure 4).

The western blot gives information on the quantity of the red fluorescent proteins and beta-actin proteins inside the cell. All the wells had the same size of beta-actin bands (Figure 5). Only wells 1,2,4, and 5 had red fluorescent bands (Figure 5).

Discussion

Based on the results of the staining (Figure 3), BCA assay standard curve (Figure 4), and the western blot (Figure 5) the null hypothesis is correct as CRISPR CAS9 was able to insert a gene into a cell. The staining process was a verification process to view the red fluorescent proteins under the microscope. Only the HeLa cells that were transfected successfully with CRISPR CAS9 will show RFP. This was because the template plasmid transfected into the HeLa cells coded for the red fluorescent proteins.

The BCA assay standard curve shows the concentration. A higher protein concentration means that the cells are acting differently which is a sign of a new gene. The western blot of RFP in the CRISPR vs non CRISPR cells shows that red fluorescent protein was detected in the CRISPR cell. All the wells had the same size of beta-actin bands which means that all the HeLa cells had roughly the same amount of actin proteins (Figure 5). The wells without any bands in the RFP means that these cells didn’t get treated with CRISPR or transfection didn’t happen successfully (Figure 5) The wells with bands in the RFP mean that there are red fluorescent proteins and transfection happened successfully.

With the success of CRISPR CAS9 it can be used to understand and possibly treat diseases. Certain diseases match with specific regions in the DNA sequence. This can be treated by formatting CRISPR to cleave those specific regions and inserting new information that will have a positive result on the organism. Another use of CRISPR is biotechnology which will make certain crops have the ability to adapt to the environment they are in.

As technology evolves researchers will find ways to use CRISPR and other genome editing tools to enhance the lives of organisms on this planet.

Appendix

Figure 1 | Plasmid Map. This image shows the guide plasmid and template plasmid that were used in the bacterial transformation. Both plasmids have the ampicillin resistor in order to allow growth in the ampicillin plate. In the guide plasmid: AAVS1 is the gene is site where the plasmid will be inserted, CAS 9 is the protein that cuts at the gene site causing a double stranded break.

Figure 2 | PCR Verification. This image shows the gel electrophoresis after the PCR step for the two plasmids. The thick bands verify that the plasmid was taken up by our bacteria. The thick fragments have a size of 1.3kbs.

Figure 3 | Staining. This image shows the morphology of normal HeLa cells and the HeLa cells with CRISPR. The DAPI stain shows the AT rich regions of DNA in the HeLa cells. The phalloidin stain shows the actin of the HeLa cells. The red fluorescent proteins are only seen in the HeLa cells that were transfected because the inserted plasmid coded for the RFPs. The merged pictures show all parts of the cell with the stains. The PMT served as the negative background control for all the microscopy images.

Figure 4 | BCA Assay Standard Curve. The BCA curve provides a linear model to determine the protein concentration. The equation is y=2573.3x-288.51 and the absorbance of our sample is 0.3220. The protein concentration of our sample is 540.0926 ug/mL.

Figure 5 | Western Blot. All the wells had the same amount of the beta-actin protein. Well #1 had the most amount of the red fluorescent proteins. Well #3 doesn’t have any red fluorescents protein therefore it must have been untreated HeLa cells. The wells with RFP mean that CRISPR successfully edited the genome of these cells.