Pglo Transformation Experiment

Abstract In this lab of transforming bacteria that was experiment today, I will be identifying the process of bacterial genetic transformation and how to calculate transformation efficiency. The samples that will be used in today’s bacteria will contain samples of E.coli sand inserted DNA plasmid into their genetic sequence. If done correctly the results will show a successful genotypic and phenotypic mutations, which will display fluorescent under ultra-violent lights or show signs to being resistant to ampicillin. This experiment was primarily for the purpose of growing E. Coli bacteria.

The color of the bacteria was a whitish color and the colony size is similar both before and after the transformation. The best way to do it is to compare the control of the experimental plates. Cells that were typically not treated with the plasmid could not grow on ampicillin, although cells that were treated with the plasmid can grow on the LB/AMP plate. The plasmid would have to confer resistance to ampicillin. Moving on, the GFP gene is what is glowing in the plate because it was activated by the sugar arabinose. The sugar arabinose and the plasmid DNA are also needed to be present because that is what initially turns on the GFP gene which makes the bacteria glow. Organisms can also turn on and off particular genes for camouflage reasons. An organism would benefit from turning on and off certain

The objective of this experiment was to genetically transform E.coli cells to express ampicillin resistance and to produce the green fluorescent protein by using the pGLO plasmid. It was hypothesized that only the cells with pGLO DNA added to the solution would be able to survive in the same environment as the ampicillin, and that only the cells grown in the plate with arabinose would fluoresce bright green. The results supported the hypothesis, showing that the +pGLO LB/amp/ara plate was the only plate that had fluorescent cells and grew. Additionally, that +pGLO cells were the only ones that grew on an LB/amp plate.

In preparing for the bacterial transformation, DNA plasmid is introduced into the E. coli cells that will express newly acquired genes. Two tubes were used and labeled both as +pGLO and -pGLO. A solution of (CaCl2) was transferred 250 µl onto the two tubes. The tubes were placed on the ice. A sterile loop was then used to gather a single colony of bacteria from a starter plate. Now, that both tubes contain bacteria they were placed on the ice for 10 minutes. Four agar plates were labeled as: +pGLO LB/amp, +pGLO LB/amp/ara, +pGLO LB, -PGLO LB/amp. Heat shock was used to transfer both the +pGLO and -pGLO, at exactly 42°C. Time was observed for 50 seconds and quickly return the tubes to the ice for another 2 minutes. As the tubes, cold down they

The purpose of this lab is to use genetic engineering to transform E. coli bacteria by inserting the plasmid pGLO, and to then see if the bacteria was transformed by using the antibiotic, ampicillin.

Next, each tube was opened and 250 µl of transformation solution was added to each and then placed in the ice. While the tubes were on the ice, a sterile loop was used to pick up to 2-4 large bacteria colonies of bacteria from the starter plates and immerse to both +pGLO. The tubes were then placed back on the ice. Once the colonies of bacteria were well placed in the +pGLO solution, the same process was repeated for the –pGLO tube using a new sterile loop.

This pBlu lab had for purpose to present the changes of the strain of E. coli bacteria due to new genetic information being introduced into the cell. In this experiment we are freezing and heat shocking the E. Coli bacteria that is then forced to take the plasmid DNA. The E. coli then transforms the pBLu plasmid, which carries the genes coding for two identifiable phenotypes. After following the Carolina Biological steps our lab worked well and we able to see some colonies of bacteria on the plates. The x-gal plate showed a significant amount of bacteria to confirm that the pBlu plasmid took over the E. coli strain.

In the pGLO Bacterial Transformation lab, Escherichia coli is transformed with a gene encoding green fluorescent protein by inserting a plasmid containing the GFP gene, beta-lactamase, and arabinose into the bacterium. Successfully transformed bacteria will grow in the presence of ampicillin and glow a bright green color under ultraviolet light. The sugar arabinose is responsible for switching on the GFP gene in the transformed cells, without it, the gene will not be expressed.

In this experiment the objective was to transform E. coli with the pGLO plasmid and calculate the transformation efficiency. The hypotheses were that the plate with only LB agar and untransformed E. coli would grow a lawn; the control plate of untransformed bacteria with LB and ampicillin would experience no growth; the transformed plate with just LB and ampicillin would grow colonies of bacteria but it would not glow green under UV light; and the transformed plate with LB, ampicillin and arabinose would grow colonies that would glow green under UV light. The results found supported each of these hypotheses as the bacteria grew as predicted. The

This experiment was performed to assess the efficacy of genetic transformations on bacteria via plasmid DNA coding for ampicillin resistance and green fluorescent protein. Genetic transformation was studied by taking transformed and untransformed Escherichia Coli (E. coli) and placing them on various media to observe gene expression via growth and color under UV light. The transformed E. coli were able to grow on ampicillin while the untransformed E. coli, which lacked the plasmid genes for ampicillin resistance, only grew on nutrient broth. In the presence of arabinose, the transformed E. coli glowed green. These results support the previous scientific understanding of bacterial competency, vectors, and gene expression and support gene transformations as an effective method to transfer the desirable DNA of one organism into another organism’s DNA. These results can be applied to real world issues such as medical treatments, food production, and environmental conservation.

The transformation of E. coli using plasmid DNA was a success. The positive control plate had a near lawn of blue colonies growing on the plate. This indicated that the E. coli cells took up the plasmid and became ampicillin resistant. The blue colonies formed because the cells were able to produce β galactoisdase and in presence of X-gal the colonies turned blue. There were light blue colonies formed near the edge of the plate. This could due to the lower concentration of X-gal near the edge of the plate so those colonies were not really blue. In addition, there were too many colonies to count so we estimated the transformation efficiency of the positive control to be around 2000 units/μg. On the other hand, the negative is shown in figure

The plate with the bacteria that is most like the original non-transformed E. coli colonies is the LB/-pGLO plate. The bacteria could grow because there was no ampicillin on the plate, and it couldn’t have been transformed because no pGLO was added. Since the plasmid wasn’t added and the environment was ampicillin free, the bacteria on the LB/-pGLO plate should be pretty much the same as the original E. coli because nothing was done to it.

There were two controls in place during this experiment. The first control was the –pGLO LB plate; which showed that without inserting a plasmid into the bacteria, that bacteria would not be able to glow. The second control was the –pGLO LB/AMP plate; which showed that bacteria that did not contain the plasmid containing the ampicillin resistant gene could not survive in an environment with ampicillin preasent. This experiment had two constants; which were, that we incubated all of the bacteria cultures at the same temperature and the fact that we used the same type of bacteria for all of the plates. A potential sources of error in this lab is that the bacteria could have gotten contaminated from being exposed to air and debri falling into the plate.

While this experiment was done very carefully, there were a few errors that were made during the experiment. For instance, while a group member was putting the transformation fluid into the microcentrifuge tubes, they accidentally tried to obtain the fluid without a sterile tip and this could have possibly had the micropipette obtain the wrong amount of fluids afterwards. Also, when retrieving the E. coli colony, a group member scooped up an entire colony instead of half of it, so there were two different bacterium colonies used in this

A transformation was performed so a vast quantity of the recombinant plasmid DNA might be obtained. There was a total of six transformations performed from four DNA ligations (excluding the fifth ligation). All six transformations were then incubated on ice for five minutes, followed by a heat shock, which came from putting the transformations in a 42-degree Celsius heat block for two minutes. Afterwards, the transformations were incubated on ice for another two minutes, then placed in a 37-degree Celsius incubator overnight, then plated. All the transformations included E.coli and luria broth, but differed in their other growth mediums. The first transformation consisted of the 1:1 molar ratio of pET-41a(+) to egfp ligation, kanamycin and