A Study On Green Fluorescent Proteins

The GFP gene is normally isolated from the jellyfish Aequorea victoria, allowing it to fluoresce with the absorption of ultraviolet light (Goodsell, 2003). This gene is inserted into a region of a plasmid to be regulated by an operon (Brown, 2011). In the plasmid, GFP is normally repressed but when arabinose is present, it will induce the gene to make enzymes able to digest the arabinose, and once there is no more arabinose,

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 plasmid pGLO contains an antibiotic-resistance gene, ampR, and the GFP gene is regulated by the control region of the ara operon. Ampicillin is an antibiotic that kills E. coli, so if E. coli, so if E. coli cells contain the ampicillin-resistance gene, the cells can survive exposure to ampicillin since the ampicillin-resistance gene encodes an enzyme that inactivates the antibiotic. Thus, transformed E. coli cells containing ampicillin-resistance plasmids can easily be selected simply growing the bacteria in the presence of ampicillin-only the transformed cells survive. The ara control region regulates GFP expression by the addition of arabinose, so the GFP gene can be turned on and

Green fluorescent protein (GFP) comes from the jellyfish Aequorea Victoria is rare proteins with high fluoresce and absorbance. The purpose this experiments is to purify and express a His2-tagged recombinant from of GFP (rGFP) from the E. coli strain BL21(DE3)< pRSETA-GFPUV > through a series of experiments by using Ni+2 agarose affinity chromatography technology. The GFPuv gene (UV-optimized GFP) was over expressed in the E. Coil strain BL21 (DE3) (pLysS) as an n-terminal His6/Xpress epitope tagged bind protein. Then using Ni2+ Agarose affinity chromatography to obtain purification of the crude extract. Then observe under the long wavelength UV light, the activity of the rGFP in the column fraction. Bradford assay was performed to obtain the total protein amount. When calculating the

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 gene for GFP is regulated by an inducible repressor system, meaning it is turned on by environmental factors. In this case, GFP is expressed when exposed to the sugar arabinose; moreover, the organism will glow only when arabinose is present. Without arabinose, the repressor attaches to the regulator/ operator segment of the gene and acts as a roadblock, preventing RNA polymerase (RNAP) from reading the structural gene and creating a complementary mRNA strand; thus, GFP protein production is prohibited. However, when present, arabinose binds to the repressor, changing its active site, so it can no longer bind to the operon; RNAP can then initiate the transcription of the GFP gene by binding to the promoter segment. Thus, I predict that the E. coli will glow only when pGLO and arabinose are present because arabinose activates the expression of GFP, a gene found in pGLO. Regulation of gene expression is crucial to prevent wasteful overproduction of unneeded proteins and to allow adaptation to new environments (Regulation of

Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).

Coli. To be able to activate the GFP gene the presence of the sugar arabinose is required. In order to make the E. Coli grow, the presence of ampicillin, an antibiotic, and pGLO is present, however, if ampicillin is present without pGLO it will stop the growth of the E. Coli because the pGLO would not be present to stop the antibiotic. In the experiment E. Coli was tested with the different proteins and plasmids, testing if the plate would glow or grow. If the E. Coli was tested with ampicillin, pGLO, and arabinose the plate would both glow and grow because of the proteins present, however, if the E. Coli was tested with ampicillin and pGLO the plate would neither glow or grow because the proteins were not

In this lab, we will perform genetic transformation to E. coli. Transformation is an insertion of a gene into an organism in order to change or add certain traits to the organism. Genetic transformation is widely used in the biotechnology field, for examples: In medicine, gene therapy involves transforming a sick person’s cell with healthy copies of the defective gene that causes the disease. In research, bacteria are transformed with genes encoding human proteins for Bio-manufacturing production or for further study of these proteins. Today’s lab, we are transforming bacteria with a gene that code for green fluorescent protein (GFP). This gene originally came from the bioluminescent jellyfish Aequorea Victoria. GFP is the

My hypothesis is, with the combination of Escherichia-coli(E-Coli), plasmid, arabinose, and a UV lamp, the E- Coli will grow and glow in the dark.

The plasmid was then inserted into a strain of E. coli and grown. After incubation the E. coli strain was found to fluoresce upon illumination with UV light inside of the growing cell, without the addition of A. victoria products (figure 1). Partially purified GFP protein from the bacteria was then obtained and found to posses an identical excitation and emission spectrum to the original A. victoria GFP, as seen in figure 2. This result suggested that GFP did not require additional enzymes/cofactors from A. victoria to fluoresce. Chalfie, using the same technique for GFP insertion in E. coli, inserted the gene into C. elegans under the control of the mec-7 gene, which encodes β-tubulin in C. elegans’ six touch receptors. The GFP again fluoresced when exposed to blue light, but this time the fluorescence was limited to the β-tubulin transcribed by the mec-7 gene, as seen in figure 3. Martin Chalfie, from the results obtained during his experiments was able to conclude that GFP isolated from A. victoria could fluoresce in living cells when exposed to blue light, without cofactors or enzymes form A. victoria. He also showed that gene expression could be traced using GFP, by measuring different fluorescence levels in C. elegans larvae as they

The main objective of this experiment was to attempt bacterial transformation, and alter the initial plasmid of DNA to one that expressed GFP, and was therefore able to glow under the UV light. The methodology we used to complete this process was bacterial transformation which is a widely used method where foreign DNA is introduced into a bacterium, which can then amplify, or clone the DNA. Our results were that there was growth on all of the plates except for the-pGLO plate with LB and ampicillin. The results for the fluorescence of the plates was that only the +pGLO plate with LB, ampicillin and the arabinose sugar glowed.

The bacterium being observed was E. coli, which codes for green fluorescent protein. E. coli is a bacterium that normally lives in the intestines of humans and animals alike, and can cause infection. One hopes to observe a glow from a green fluorescent protein (GFP) which is typically made by a jellyfish. This will be made possible by the additional presence of arabinose, which acts as the inducing agent. The inducing agent influences the transcription of the GFP and activates the transcriptional promoters. A promoter allows a foreign gene to fall into place much easier resulting in gene expression. It is expected that the combination of the plasmid, with the Lysogeny Broth (LB) and the arabinose (ARB) will transform the bacteria, and allow for growth of the bacteria in the presence of the antibiotic ampicillin (AMP) along with an observable glow under ultraviolet (UV) light.

The aim of the experiment was to observe the genetic transformation and determine the transformation efficiency of the bacteria, E.Coli.

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.