Pglo Transformation Lab Report

Abstract: Bacterial transformation involves the change of genetic composition of bacteria by altering its genetic identity. The pGLO plasmid was ingrained in the E. coli cell, which allows the modified E. coli cell to begin to code for the GFP protein gene and the beta lactamase gene (ampicillin resistance gene). After modifying the bacteria cell, the changes involved with the plasmid were tested on 4 plates, two plates containing the pGLO plasmid (+) were treated with LB nutrient media. One of the LB plates contained arabinose in it, which should fluoresce green under UV light. The other LB plate contained ampicillin. Two other plates which did not contain the pGLO plasmid (-) both had LB media growth, one plate just to show cell growth,

and the other plate was to show that the ampicillin killed off the cells. Concluded from the results was that the cells containing the pGLO gene could grow on plates containing ampicillin, and that the plate containing arabinose would fluoresce green.
Introduction: The transformation of bacteria is a process by which DNA is transferred to a new cell.

Through transcription and translation, the DNA (plasmid) from one organism (jellyfish pGLO) can be expressed in another organism (E. coli). Plasmids are used for this experiment due to their small size, making it easier to enter the bacteria.
Transformation of bacteria can happen by two different procedures, electro-puration, or electro-compentent. Electro-puration was the method used, it involves heat shocking the cells with calcium ions. This was the method used because it left the most amount of viable cells as compared to other methods.
The plasmid pGLO contains the GFP gene which can only be activated while in the presence of arabinose. The plasmid also contains an ampicillin resistance gene, which explains why bacterial cells transformed with pGLO can survive when ampicillin is present. GFP is a protein that is found in jellyfish, and is known for emitting a green fluorescent light. In vitro, it is able to fluoresce this green color by releasing photons from being in an excited energy state. The energy was provided by a UV light at the end of this experiment. Jellyfish can fluoresce this green on their own because in vivo, GFP works with another protein that allows the GFP to fluoresce without being in an excited energy

state.
Ampicillin was used because it has the ability to kill E. coli cells. Ampicillin resistance is another gene present in pGLO, therefore to test if the transformation occurred properly, the cells that were in the presence of ampicillin would survive. The resistance gene codes for an enzyme called beta-lactamase. (Mosher 2002). Another gene carried by pGLO is ARAC which binds to arabinose and creates a transcription factor. When in the presence of arabinose, pGLO will fluoresce.
Sodium dodecylsulfate-polyacrylamide gel electrophoresis is a method used for the separation of proteins. It separates molecules based on the size of them through an electric field. Different buffers were used so the gel electrophoresis could be carried out properly. Laemmli sample buffer contains a tris buffer that hold the pH of the bacteria at 6.8, glycerol, SDS (detergent) or sodium dodeclysulfate and DTT or dithiothreitol. Bromophenol blue is also added to track the protein in the gel. The buffer is used to denature the proteins from their “larger” shape and into a smaller string of amino acids. Denaturation of these proteins is caused also by heat. The heat causes the breakage of Vander walls interactions, hydrogen bonds and covalent bonds. The breakage of bonds allows the proteins to return to primary structure.
Materials and Methods: E.

coli cells were grown at 37 degrees Celsius with calcium ions. These cells were then incubated in ice and placed into 4 polypropylene tubes. Each tube consisted of 25 microliters of E.coli cells. Two of the four were labeled with (+) while the other two were labeled (-). Positive was with samples that contained 100 pg/ microliter of pGLO, while negative was for the samples that did not. The 4 tubes were incubated in ice for 30 minutes, then heat shocked for 30 seconds at 42 degrees Celsius. The 4 tubes were then returned to the ice bath for 5 minutes. After the removal, .975 microliters of LB media was added to each tube at room temperature. The tubes were then shook at 225 RPM at 37 degrees Celsius for one hour. 300 microliters of cells were then extracted from the tubes and then placed in agar plates. The cells were spread across the plate and the incubated overnight at 37 degrees

Celsius.
After overnight incubation and after being examined under UV light, the four plates with the E. coli cell samples were harvested and mixed with the Laemmeli’s sample buffer. After, two of the tubes were heated. A sample was extracted from all of the tubes and inserted in the gel wells along with molecular markers. The gel contained 4-20% Tris-HCl, 10 wells, and each 30 microliters. The gel was surrounded by a detergent buffer (100 mL buffer, 900 mL water), with a final concentration of 1x solution is 25mM Tris, 192 mM glycine, and .1% SDS. Once the buffer was in the electrophoresis box, it was hooked up to an electrical current. After 30 minutes, the gel was moved into water for 5 minutes, with changing the water 3 times after every 5 minutes passed. Then the gel was placed in kamasi blue and spun for a week. After a week, the gel was examined under UV light.