Genetic Transformation Experiment Report

Categories: Biology

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Abstract

Genetic transformation is a process in which foreign genetic material is introduced into an organism, leading to the acquisition of new traits. In this experiment, we investigated the ability of the plasmid pGLO to transfer the Green Fluorescent Protein (GFP) gene from a jellyfish into Escherichia coli (E. coli) bacteria. Four different plates were used to assess the transformation: a control plate with only LB agar, a plate with LB agar and ampicillin, a plate with LB agar, ampicillin, and pGLO, and a plate with LB agar, ampicillin, pGLO, and arabinose.

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The presence of arabinose acted as the switch to activate the GFP gene.

Introduction

Genetic transformation involves the incorporation of foreign genetic material into an organism, resulting in a change in its genetic makeup. In this experiment, we focused on the use of the pGLO plasmid to transfer the Green Fluorescent Protein (GFP) gene from a jellyfish into Escherichia coli (E. coli) bacteria. The successful transfer of the GFP gene would lead to the expression of a fluorescent trait in E.

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coli, allowing us to assess the effectiveness of pGLO as a vector for gene transfer (Federoff and Wagner, 2014).

E. coli is considered competent when it can take up foreign DNA, making it a suitable candidate for genetic transformation (Weedman). To verify the transformation's success, we employed four different plates. Three of these plates contained ampicillin, an antibiotic to which pGLO is resistant. Ampicillin was used to eliminate E. coli cells that did not acquire the pGLO plasmid. The fourth plate contained arabinose, serving as the activator for the GFP gene, resulting in a fluorescent phenotype (Weedman).

Materials and Methods

In this experiment, we followed a set of procedures to assess the genetic transformation of E.

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coli using the pGLO plasmid. The following steps were performed:

Wearing vinyl gloves to ensure safety, label two microcentrifuge tubes as "+pGLO" and "-pGLO."
Using a micropipette, transfer 250 μl of the transformation solution to both centrifuge tubes.
Place both tubes on ice for ten minutes.
Using a sterile loop, introduce a single colony of E. coli bacteria into both tubes.
In the "+pGLO" tube, add the pGLO plasmid.
Return both tubes to the ice for another ten minutes.
Perform a heat shock by immersing both tubes in a 42°C water bath for 50 seconds, followed by immediate transfer back to the ice bath for two more minutes.
Pipette 100 μl from each tube onto their respective plates according to the labels ("+pGLO" on the "+pGLO" plate and vice versa).
Spread the liquid evenly on each plate using separate sterile loops for each plate.
Label the plates and stack them for incubation at 97°C for subsequent analysis (Bacterial Transformation 2013).

Results

The results of the experiment are summarized in the following table:

Plate	Components	Colonization	Fluorescence
1	-pGLO LB	Yes	No
2	-pGLO LB/amp	No	No
3	+pGLO LB/amp	Yes	No
4	+pGLO LB/amp/ara	Yes	Yes

The results show that Plate 1, which served as a control with only LB agar, allowed E. coli to grow but did not exhibit fluorescence. Plate 2, containing LB agar and ampicillin, did not support colonization, as ampicillin killed E. coli without the pGLO plasmid. Plate 3, with LB agar, ampicillin, and pGLO, allowed E. coli to colonize but did not exhibit fluorescence. Plate 4, which contained all necessary components, including arabinose, demonstrated both colonization and fluorescence, indicating successful genetic transformation (Bacterial Transformation 2013).

Discussion

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.

Conclusion

In conclusion, this genetic transformation experiment confirmed that the pGLO plasmid can transfer the GFP gene from a jellyfish into E. coli bacteria when all essential components are provided. Despite challenges with plate preparation and incubation, the successful colonization and fluorescence observed in Plate 4 validate the feasibility of genetic transformation. Improvements in plate handling and observation could enhance the accuracy of future experiments.

Recommendations

Based on the challenges encountered in this experiment, several recommendations can be made for future studies:

Implement stricter control measures during plate preparation to minimize contamination and ensure accuracy.
Pre-label plates to reduce the risk of mix-ups and labeling errors.
Optimize the incubation process to allow for periodic observations and minimize external disturbances.
Consider using alternative techniques or materials to address issues with plate denaturation.