How The Genetic Transformation And Determine The Transformation Efficiency Of The Bacteria

Over the course of this lab, our class performed a transformation on bacteria, altering its DNA to cause it to fluoresce when exposed to UV light. Transformations, the process of taking in foreign DNA, are common procedures in the field of biotechnology, the exploration of life processes to develop new technologies. Biotechnology applies recombinant DNA technology, which is DNA made from two or more organisms, to improve organisms and solve issues. Scientists use plasmids, circular pieces of DNA, to “cut and paste” genes into other organisms. Researchers often use GFP, or green fluorescent protein, in their experiments and procedures. GFP naturally occurs in jellyfish causing them to glow green under UV light. Scientists worked with GFP when designing GloFish. Their original plan was to create a fish that could signal pollution in water, but they ended up developing a beautiful, glowing pet fish which is now sold throughout the U.S. We made

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

The purpose of the PGLO lab was to be able to perform a procedure known as a genetic transformation. We used a procedure to transform bacteria with the gene that codes for a Green Fluorescent Protein (GFP). The actual source of the GFP gene that we used in this complicated experiment is the bioluminescent jellyfish Aequorea victoria. This protein causes the jellyfish to glow under a UV light that was provided in the dark. After the transformation procedure, the bacteria showed their newly acquired gene from a jellyfish and produced the fluorescent protein, which as a result, causes it to glow. If the bacteria glowed in the dark, that was the initial sign that the experiment was successful.

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.

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.

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

One of the most imperative functions in maintaining the development of evolution is the frequency of genetic transformation: the injection of foreign DNA into another organism’s DNA. This term is defined by the actions of a vector, but more specifically by the actions of plasmids and phages. However, in this experiment we are primarily focused on the effect of the pGLO plasmid transformation of GFP on the E. coli bacteria by introducing a second chromosome or a plethora of cloned plasmids. (Bassiri 2011)

The field of biotechnology involves the concept of genetic engineering, altering the DNA/genetic material of an organism using information from a different one. The process in which bacteria can obtain this manipulated genetic information from another source is called genetic transformation. The goal of this experiment was to genetically transform Escherichia coli bacteria’s DNA by inserting the vector pGLO plasmid which codes for ampicillin resistance as well as the green fluorescent protein, GFP. For the experiment, the E. coli bacteria were separated into two groups; control and

In our hypothesis we stated that only the container containing all of the components +pGLO, LB broth, ampicillin, and arabinose would be the one that genetically transformed. In order for the bacteria to grow at a rapid pace all it needed was LB broth but when you added ampicillin, an antibiotic, it killed off all of the bacteria. +pGLO has the gene to resist the antibiotic so when that was added it was allowed to grow but there was no sugar to turn on the glowing protein. Finally, after arabinose, a sugar, was added it turned on the switch located in the +pGLO for the fluorescence and enabled to grow and glow.

Genetic transformation occurs when genes are inserted into another gene to change the organism’s trait (Weedman2016). In this experiment, we proceeded to transform the E. coli bacteria with a gene that contained green fluorescent protein. The green fluorescent protein is used in experiments because it beams a green color under a UV light (Chalfie2008). Typically, it is used to mark the expression of genes, which is why it serves as the symbol for all gene expressions (Tsien1998). In the experiment, we will be using pGLO as the organisms that will transmit the disease, otherwise known as a vector. The pGLO in the experiment

coli, to express a gene that is normally causes jellyfish to be fluorescent. The plasmid that contains the gene for the green fluorescent protein (GFP) is called pGLO. The type of genetic transformation we used in the experiment was heat shock which made the membrane of E. coli fluid and allowed the pGLO to enter the cell. In order for the E. coli to express the GFP gene there needs to be arabinose presence, which is a sugar that acts as an on switch for the pGLO. Using this information, we predicted the bacteria E. coli will grow and express the GFP on the plate that consists of +pGLO, ampicillin, arabinose, and LB broth (Luria Bertani broth).

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 discovery of the gene transfer mechanisms could be attributed by the work of Lederberg and Tatum back in 1946. Using Escherichia coli(E.coli) as their model, they proposed the genetic materia of E.coli could be exhanged via sexual process. In order to prove their hypothesis, they mutated 2 wild type E.coli strains(K12) using X-ray or ultra-violet radiation to produce Y-10 and Y-24 mutant strains. The former was auxotrophic to threonine, leucine and thiamin whereas the latter failed to produce biotin, phenylalanine and cystine[1]. These mutant could only survive in mininal media plates provided that the aforementioned amino acids are supplied accordingly to each mutant strains.

The first step of this experiment was to determine if genetic transformation has occurred within the E. coli culture. The plates that were compared in this section can be seen in Figure I. The plates that were compared were labeled LB+ amp (+) and LB + amp (-). The positive control was the untransformed cell in LB+ amp(-). This control was testing: if the antibiotic would kill all of the untransformed cells in the LB+ amp (+). In figure I. the plate labeled LB + amp (-) had no bacteria on it. Therefore, the antibiotic did kill all the bacteria that did not transform. In conclusion, the plate labeled LB+ amp (+) contained only transformed cells, and this can be seen In figure I., because the bacteria on the plate labeled LB+ amp (+), are

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.