Effects Of Double Homologous Recombination

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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. …show more content…
Synechocystis does this by double homologous recombination and in this case, the process was manipulated by making the plasmid pKCP43 and disrupting the psbC gene. Since the two ends of the plasmid and the gene are extremely similar, the Synechocystis bacteria can easily uptake the plasmid leaving behind the psbC gene during crossover in homologous recombination. As the bacteria replicate the kanamycin resistant gene present in the pkCP43 plasmid, continue to reproduce while the bacteria that did not contain the mutation are eventually killed by increasing concentrations of kanamycin. Our colonies were provided with BG -11 and glucose to serve as the nutrient medium for growth because the psbC (necessary for photosystem II) gene is knocked out of Synechocystis with the replacement of the KanR gene introduced via the pKCP43 plasmid. Initially, the Synechocystis bacteria had to be plated without any Kanamycin to allow it to grow since the replication rate for this particular bacteria is about twelve hours. We expected a fraction of the bacteria colonies to successfully transform and to our advantage, new colonies that successfully underwent double homologous recombination began to emerge as shown in figure 3. We continued to add kanamycin in the following week to ensure that the modified organisms continued to grow while the incompetent …show more content…
After resuspension with TE buffer our aggregate DNA was ready for PCR amplification, gel electrophoresis, and UV screen. The white colonies shown in Figure Eight comprised the psbC gene and our methodology proved successful with the UV analysis displayed in Figure Nine. The results revealed that the plasmids containing necessary gene was successfully process in agreement with Figures Eight and Nine. Next, our synechocystis transformants were segregated into increasingly higher concentrations of kanamycin for further selection because synechocystis maintains multiple copies of its genes. By the eleventh week we were ready to subject our transformants to the processed plasmid from week nine. We employed the use of bead vortexing to disintegrate the E. Coli cell wall and extract and reintroduction of psbC gene the containing plasmid for spontaneous uptake with synechocystis. After recombining and growing over the course of one week after plating we observed growth of suspected transformed synechocystis. Figure Ten shows the replated cells onto the new growth medium lacking glucose. Referring to Figure Eleven, we saw that the synechocystis transformation successfully took the plasmid, recombined, and expressed the phenotype originally found in the wild type species. The species on the right The psbC- species are transformed to carrying the psbC gene once again

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