Pglo Plasmid Experiment

The goal of this experiment was to investigate genetic transformation of E.coli through the reaction of organism to the vector pGLO plasmid. As mentioned, the pGLO plasmid contains genes coding for resistance to ampicillin (amp), and genes coding for production of the green fluorescent protein (GFP) which glows under UV light in the presence of arabinose (ara),which serves as a reporter gene. This green fluorescent

The pGLO plasmid will transform the E. coli bacteria with a gene called GFP that codes for the Green Fluorescent Protein in the genetic code. GFP was discovered in the jellyfish, Aequorea victoria as a green fluorescent light emitted from the jellyfish. It was typically seen in the dark upon its activation and since then has been used in studies relating to genetic transformation. (Chalfie and Tu 1994) The majority of the studies test the many different factors that are required in the transformation of pGLO which will determine the functionality of GFP in the E.coli bacterium. The first experiment in transforming GFP and E. coli was completed in 1994 by Chalfie and was further refined the same year. The experiment proved the importance of using restriction enzymes, and DNA ligase in the process of transforming GFP to identify arabinose as the primary activator, and to identify the ampicillin

The purpose of this experiment was to show the genetic transformation of E. coli bacteria with a plasmid that codes for Green Fluorescent Protein (GFP) and contains a gene regulatory system that confers ampicillin resistance. A plasmid is a genetic structure in a cell that can replicate independently of chromosomes. In this lab, the Green Fluorescent Protein, which is typically found in the bioluminescent jellyfish Aequorea Victoria, was cloned, purified, and moved from one organism to another with the use of pGlo plasmids. It was hypothesized that if bacteria that were transformed with +pGlo plasmids are given the gene for GFP, then transformed cell colonies

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.

Tsien, Roger Y. "THE GREEN FLUORESCENT PROTEIN." Annual Review of Biochemistry 67 (1998). Howard Hughes Medical Institute. Web. 7 Nov. 2016.

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.

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 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

Purpose: The purpose of this experiment is to teach the students step by step on how the genetic transformation process works using pGLO. Genetic transformation is when an individual cell’s genetic material is changed by exogenous DNA.

Genetic transformation happens when a cell is forced to take up an outside piece of DNA, from another living organism (Weedman 2015). A competent is a cell that is able to take up foreign genes during transformation. When this happens though the organism accepts one or more genes which then can change how it looks, can change the traits. An example of this would be in our current time with medicine, when a person has old or destroyed genes we then can remove the parts of the DNA that are effected and inject or replace them into the body with healthier ones (Weedman 2015).

This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.

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.

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

The purpose of this study was to see whether E. Coli cells would engage in the pGLO plasmid and glow in the presence of four control environmental factors which are arabinose sugar, bacteria, the antibiotic ampicillin, LB nutrient broth and pGLO plasmid DNA. This was tested using four plates, all the plates had E. Coli cells and different environmental factors. The founding was that E. Coli will only fluoresce when bacteria, pGLO plasmid DNA, the antibiotic ampicillin, and LB nutrient broth are present. The result did not support the hypothesis because it stated that, E. coli cells that are exposed to the pGLO plasmid would engage in the plasmid and glow only if the arabinose sugar is present.

In this lab, we demonstrated that a plasmid could be inserted into the DNA of bacteria in order to alter the physical characteristics of the bacteria. We used biotechnology to manipulate the genetic code of the bacteria so that it would glow when exposed to a UV light. After a couple of days when the glow of the bacteria started to fade, it became apparent that the operon system that enabled the pGLO gene to take affect was stopping.