Purpose: The purpose of this experiment was to oversee the process of genetic transformation. We were trying to see how this procedure accounts for the pGlo plasmid's ability to code for Green Flourescent Protein (GFP)
Introduction: In this lab we were to perform a procedure known as genetic transformation. This protein gives the organism a particular trait. This involves the insertion of a gene into an organism in order to change the organisms trai. We used that procedure to transform becateria with a gene that codes for GFP. The source of this particular gene is from the bioluminescent Jellyfish Aequorea Victoria, which causes them to glow in the dark. Once the procedure is completed, the acquired jellyfish gene will cause the bacteria to glow a green color under ultraviolet light. In this experiment, we were to learn about the process of moving genes from one organism to another with the help of a plasmid. A plasmis contains genes for one or more traits needed for its survival. Transferring of said plasmid occurs between bacteria to allow them to share these beneficial genes and hence, the bacteria adapts to its new environment. In this experiment, the occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids. The pGlo plasmid encodes the gene for GFP and the gene for resistance to the anitiotic ampicillin. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells nutrient medium. Transformed cells will appear white without the sugar, and glow green with the arabinose.
Methods: At the start of the lab, we labeled 4 tubes with either +PGlo, -PGlo, (control group), transformation solution, and broth. Then we pipettes some transformation solution to both the negative and positive tubes and then, we put them in ice. After that, we scooped bacteria out of a Petrie dish and spread the E. Coli in both with a sterile loop and mixed well. However, we put plasmid only in the positive pGlo container. In order to compare results, we labeled 4 different Petrie dishes. After, we put the tubes back in ice for 10 minutes and then we used heat shock by putting it in water (45 degrees Celsius) for 50 seconds and back in ice for 2 minutes. Using a pipette, we put broth in both tubes. Using a pipette, we put pGlo according to how the Petrie dishes were labeled as such. Then, we spread out the solution with a loop to increase surface area. The last step was to put it in an incubator at 37 degrees Celsius for 1 day. He next day, we took it out and using a UV flashlight, we measured growth in Petrie dish and it's ability to glow under the light.
Data:
Discussion: After taking it out of the incubator, we were to analyze the growth and ability of the E. Coli bacteria to glow under UV light. We had the most growth with -pGlo LB, no growth in -pGlo LB/AMP, and some growth in both +pGlo/AMP and +pGlo/LB/AMP/ARA. It was clear since before we did the lab that -pGlo LB/AMP would have no growth because all the bacteria cultures that would've grown would have been killed off by the AMP(Ampicillin) which is an antibiotic. -pGlo LB Was used as a control group because the bacteria was allowed to spread without the plasmid. +pGlo/AMP and +pGlo/LB/AMP/ARA Both had growth because the bacteria was made resistant to the antibiotic and were allowed to spread. However, +pGlo/LB/AMP/ARA was made to resist the antibiotic and the arabinose activated the pGlo gene which causes it to glow under UV light. These results are valid because we discussed what each of the Petrie dishes should have looked like. We were shown expected results of the dishes and out data was compatible. The Petrie dishes showed that the ampicillin killed the E. Coli that did accept the pGlo gene and we proved that the arabinose help the pGlo gene activate after certain genes have accepted the pGlo gene.
Conclusion: The final results of our pGlo lab yielded an effectively added Aequorea Victoria that is the bioluminescence gene found in jellyfish showed through in the Petri dish of +pGlo with LB broth, Ampecilin and Arabanose sugar. Our hypotheses proved correct because we expected the fourth Petri dish, which held every additive in it, to not only accept the pGlo gene but also to isolate it on the account that we added the Ampecilin to eliminate any remaining genes that had not accepted to bioluminescence gene. As our final results matched the expected results fairly similarly, we can conclude that the lab went relatively successful. For improvements, we could have attempted to cause greater colony growth on the Petri dishes as that would have shown more efficiency in our dishes.
