The intent of this experiment was to successfully transform synechocystis to a psbC lacking species through double homologous recombination. Subsequently we intended to retransform the mutated colonies back to the psbC carrying species after kanamycin selection. The initial knockout of the psbC gene ensured that photosystem II wouldn’t work and the colonies would rely on glucose as a source of sustainable nutrition. The sequential addition of kanamycin killed off the species that lacked the KanR gene that was replacing the psbC gene necessary for photosynthesis. Once we were content with the selection we were able to retransform the species back to the wild type carrying the psbC gene and reinstall the ability to use light as a source of energy.
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The psbC sample was loaded along with a DNA ladder to assure that the sample was within the range of the predicted size. During the PCR amplification the region in the synechocystis that gets amplified is between 662 to 2,819 bp which makes the target gene approximately 2,157 bp. We used primers to flank the psbC gene and primers to identify the presence of KanR gene. When the sample was loaded onto the gel, it separates the fragments by size, by using a marker at the 2,100 and comparing it against the samples one can see that the psbC gene is the predicted size. For the psbC gene to be visible, a loading dye was added to the sample, improper mixing of the dye and psbC sample may have caused some bands to appear brighter than the others. Another reason for this error may have been the concentration of the psbC gene present in the sample may have affected the overall brightness on the agarose gel. The reason` PCR was performed was to amplify the wild-type psbC gene and eventually reintroduce photosynthetic ability back into the Synechocystis genome. To be able to increase this particular gene CP1 forward primer at the 662 site and CP2 reverse primer at the 2,819 site as mentioned in the results these primers are used to initiate DNA synthesis while the Taq polymerase assembles the DNA strands formed while dNTPs extends the annealed primers. Purification of PCR …show more content…
This is probably due to the fact that the cells without resistances are slowly dying off. An anomaly occurs from Figure Four to Figure Five in which the colonies seem to increase. One explanation for this might be that the contamination from the previous plate (Figure 3) managed to destroy the synechocystis cells by out-competing it for resources, since synechocystis grows very slowly compared to other bacteria, there was more cell death in the plate. As stated previously in Figure 5 the cells transferred were only those that were green therefore those bacteria that grew on the plate were only synechocystis bacteria that were resistant to kanamycin and were not competing against any other contaminant and could grow
The psbC sample was loaded along with a DNA ladder to assure that the sample was within the range of the predicted size. During the PCR amplification the region in the synechocystis that gets amplified is between 662 to 2,819 bp which makes the target gene approximately 2,157 bp. We used primers to flank the psbC gene and primers to identify the presence of KanR gene. When the sample was loaded onto the gel, it separates the fragments by size, by using a marker at the 2,100 and comparing it against the samples one can see that the psbC gene is the predicted size. For the psbC gene to be visible, a loading dye was added to the sample, improper mixing of the dye and psbC sample may have caused some bands to appear brighter than the others. Another reason for this error may have been the concentration of the psbC gene present in the sample may have affected the overall brightness on the agarose gel. The reason` PCR was performed was to amplify the wild-type psbC gene and eventually reintroduce photosynthetic ability back into the Synechocystis genome. To be able to increase this particular gene CP1 forward primer at the 662 site and CP2 reverse primer at the 2,819 site as mentioned in the results these primers are used to initiate DNA synthesis while the Taq polymerase assembles the DNA strands formed while dNTPs extends the annealed primers. Purification of PCR …show more content…
This is probably due to the fact that the cells without resistances are slowly dying off. An anomaly occurs from Figure Four to Figure Five in which the colonies seem to increase. One explanation for this might be that the contamination from the previous plate (Figure 3) managed to destroy the synechocystis cells by out-competing it for resources, since synechocystis grows very slowly compared to other bacteria, there was more cell death in the plate. As stated previously in Figure 5 the cells transferred were only those that were green therefore those bacteria that grew on the plate were only synechocystis bacteria that were resistant to kanamycin and were not competing against any other contaminant and could grow