E. Coli Genetic Transformation with pGLO Plasmid Essay

Custom Student Mr. Teacher ENG 1001-04 7 April 2016

E. Coli Genetic Transformation with pGLO Plasmid

Genetic transformation is where one organism takes on a characteristic from another organism (Bacterial Transformation 2013). For this experiment we used the bacteria E. Coli to take in foreign jellyfish DNA which will allow it to change genetic material. This experiment determines the effects that the plasmid pGLO has in transferring the Green Florescent Protein found in a jellyfish into the bacteria. It determines whether or not pGLO acts successfully as a vector to move genes from one organism to the E. Coli organism (Federoff and Wagner 2014). If the E. Coli is a competent organism, meaning it allows for the uptake of foreign DNA, then the vector will successfully be able to transfer the Green Florescent Protein into the bacteria’s cells (Weedman). There are four separate plates in which we are conducting the experiment. Three of the four contain the antibiotic ampicillin which the pGLO is immune to. We use ampicillin to determine if the pGLO actually works by using it to kill off all of the cells that did not obtain any of the pGLO. Only one of the four plates contains the sugar arabinose which is needed to turn on the GFP gene (Weedman). Then there is one plate that is used as the control, and it only contains the LB which is needed for any bacteria to grow. All of the plates are necessary to prove that the pGLO actually has the proper effect on the E. Coli cells. The results of the plate with all of necessary components for genetic transformation will be the florescent effect (Weedman).

Materials and Methods:
In Weedman’s genetic transformation experiment, we attempted to determine whether pGLO was a successful plasmid to transfer GFP into E. Coli DNA. The first thing we did was obtain vinyl gloves to protect us from the E. Coli that we worked with throughout the entire lab. Then we obtained two micro centrifuge tubes and labeled one +pGLO and the other –pGLO. After, we used the micropipetter to transfer 250 (ul) to both centrifuge tubes. Right after, we closed the lids and put the tubes on ice for ten minutes. After the ten minutes were up we used a sterile loop to put a single colony of bacteria in both tubes. In the +pGLO tube we also added the pGLO plasmid. Then we put both of the tubes back into the ice for ten minutes. While the tubes were incubating, we collected our four test plates from the TA. We labeled the plates accordingly. After the ice bath, we heat shocked both tubes for exactly 50 seconds in the 42 C water. After the fifty seconds, they are transferred immediately back to the ice bath for two more minutes. Finally, 100 (ul) is pipetted from the tubes to each of the plates, following the labels. (+pGLO goes in the +pGLO plate and vice versa). After the liquid gets transferred to the plates it is then spread around using a different sterile loop for each plate. Once we complete all of the steps, we stack all of our plates, label them, and hand them to our TAs to be incubated at 97 degrees Celsius for the results in the next lab (2013). Results:

This experiment deciphered the effects that GFP had on the bacteria E. Coli with the vector pGLO. There were four different plates used to determine the correct effects. Plate 1 (-pGLO LB) was used as a control group and was only given the LB needed to properly colonized bacteria. In plate one, the E. Coli was grown. Plate 2 (-pGLO LB/amp) contained the broth needed to grow a colony as well as the antibiotic ampicillin which kills off E. Coli unless it has pGLO which is immune to the antibiotic. Because plate 2 did not possess any of the pGLO to successfully transfer the GFP, all of the E. Coli was killed off. In plate 3 (+pGLO LB/amp) there was the broth, the ampicillin, and the pGLO. Due to the pGLO being present the ampicillin did not kill off the bacteria, and it was able to colonize. The final plate 4 ( +pGLO LB/amp/ara) contained all of the components necessary to see the transformation. The arabinose acted as the on and off switch for the GFP, and as a result, showed the florescent characteristic. Below shows the results of the same experiment done in a different lab.

-pGLO LB (Plate 1)YesNo
-p GLO LB/amp (Plate 2)NoNo
+pGLO LB/amp (Plate 3)YesNo
+pGLO LB/ amp/ ara (Plate 4)YesYes

The purpose of this lab was to observe if the GFP would be transferred to the E. Coli bacteria through the pGLO plasmid. We hypothesized that only plate 4 would both grow and glow due to it being the only plate containing the necessary arabinose to activate the gene. We also predicted that plate two would be the only plate to not colonize due to it containing ampicillin but no pGLO. All of our results were right in the correct experiment; however, our plates were faulty. Our plates had been denatured after the ampicillin was added which, in turn, denatured the ampicillin antibiotic. Because of that, it did not do its job in killing any of the bacteria that had not taken the GFP gene through the pGLO. Although our plates were not successful, we were able to view some of the correct ones and observed that plate 4 did possess the florescent gene that we wanted to transfer to it. In pGLO Bacterial Transforation Kit, the results were exactly the same as the correct plates. The bacteria on the samples provided by their report seemed to have colonized more than ours, which could have been because of how the plates were created. (pGLO Bacterial Transformation Kit 2012). One major weakness in our experiment was that we had no control over the making of the different plates.

All of the plates were very easily messed up which contaminated a lot of the results within the experiment. Also, since the plates were not pre-labeled, they could have easily been mixed up or labeled incorrectly. Another weakness would be the incubation stage. Due to the fact that it took so long, we were not able to observe it. Because we were not able to observe it we were also not able to know if any outside force disturbed the incubation. Some problems that my group encountered was making sure that the correct amount of transformation solution was entered into the tubes. Another potential problem was making sure that we kept all of the different plates straight and that we put the correct solution into each plate. The results of this experiment displayed a genetic transformation where E. Coli took up the GFP gene from a jellyfish. With all of the correct material, the transformation was successful seeing as with the pGLO, the LB, the ampicillin, and the arabinose the E. Coli did indeed give off a florescent glow. Although there were various problems with our particular plates, with the correct plates, the transformation occurred just as it was supposed to.

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