Pglo Transformation Experiment

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.

E. coli HB101 was transformed with pGLO plasmid then grown on media containing ampicillin and/or arabinose and on medium containing neither (Brown, 2011). This is done for selection of transformed cells since not all cells are expected to take up the plasmid (Brown, 2011). We also expect roughly the same CFU on any plate(s) receiving samples from the same microcentrifuge tube, since they are getting the exact same

70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first

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.

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.

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.

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.

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.

The focus of this lab was to identify which plasmid (PFG or Pglo) was inserted into the E. coli culture. In order for the E. coli to be transformed, the E.coli must first be made competent. The cells were made competent by incubating the cells in calcium (2+) and then in cold temperatures. A quick heat shot opens pores in the cell membrane and allowed plasmid DNA to enter the cells. The plasmid that was inserted into the E. coli culture had antibiotic-reistance gene and a reporter gene. A reporter gene is a gene that is easy to detect, and for this lab it could be Pglo or PFG. The cells were placed in plates that contained antibiotics to kill any bacteria that did not uptake the plasmid, this is necciary because about 1% of bacteria will be transformed. This allowed only cells that up took the DNA to

In this lab the genetic process known as transformation will be used to make E. coli bacteria cells glow in the dark by adding GFP. Transformation is when a cell’s genotype is changed by adding exogenous DNA. This process results in the cell accepting the added genes and incorporating them into the reproduction process, creating more cells with the new gene. Genetic transformation naturally occurs in some species of bacteria, however in this lab it will be generated artificially (Bacterial Transformation). In order for transformation to take place the bacteria must be competent. This means that the bacterium needs to be ready to take in the added genetic information from its environment. These genes are absorbed through the cell membrane (Genetic Competence in Bacillus Subtilis). However, for transformation to occur, a restriction enzyme is required.

The two control plates LB AMP-PGLO and LB AMP+PGLO (refer to Figure 3.0 and Figure 4.0) include a nutrient causing growth and the antibiotic, ampicillin, with or without the plasmid DNA. The purpose of the two is to provide a determination of the LB AMP ARA+PGLO and whether or not, the plasmid has an effect with the sugar. In addition, the two controls show that plasmid, pGLO, can affect the growth of bacteria with the consideration of ampicillin. As well, the two control plates prove that the results of the LB AMP ARA+PGLO and the LB-PGLO plate can be reliable knowing that they have been under the same conditions of the incubator as the control plates. Green Fluorescent Protein acts as the biological molecule for the fluorescence.

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

This experiment was performed to test the hypothesis if LB nutrient broth, +pGLO and -pGLO Ampicillin, and Arabinose was placed in the E. coli plates, then there will be a significant growth in the newly transformed bacteria and it will possess the ability to glow under UV light. The measurements were recorded from the bent glass tube in each glass test tube. The transformation protocol tested for the newly possessed traits in E.coli bacteria. Throughout the experiment there were many probable reasons for failure. If the pipettes and sterile loop were not thrown out in between each use, a cross contamination could cause a miscalculation in the experiment causing the data results to fail. The hypothesis that was tested was validated due to the positive results with each experiment stating that newly transformed organisms due in fact pass on traits.