Pglo Transformation

Topics: Ultraviolet, DNA, Plasmid Pages: 5 (1825 words) Published: April 30, 2013
Connor Lauffenburger
pGlo Transformation Lab Report
I Introduction
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 will be located on the LB/amp/ara and LB/amp agar plates. Cells that have been transformed with +pGlo plasmids have the ability to grow in ampicillin plates, and the arabinose sugar allows the colonies to be visibly fluorescent under ultraviolet light. The GFP is able to resist ampicillin because of the Beta- Lactamase protein that is produced and secreted by the bacteria that have been transformed to include it in their plasmids. Arabinose is a carbohydrate, normally used as a source of food by bacteria. Bacterial colonies are not able to grow on –pGlo plates because they are sensitive to ampicillin. In this lab, I will move the GFP gene from one organism to another with the use of pGlo plasmids.

II Materials and Procedures
The first procedure of the lab was to obtain two microtubes and label one +pGlo and the other –pGlo with a marker. Next, the tubes were placed in the microtube rack and 250 μl of CaCl2 was transferred into each tube using a sterile pipette. After placing the microtubes in ice, a sterile inoculation loop was used to pick up a single colony of bacteria from the E. coli starter plate. Next, the +pGlo microtube was removed from the ice and, to ensure that the colony was efficiently dispersed into the transformation solution, the loop was gently swirled at the bottom of the tube. Once the bacteria was completely dissolved in the solution, the microtube was placed back in the ice and this procedure was repeated for the –pGlo using a new, sterile inoculation loop. From here, the pGlo DNA solution was viewed under the ultraviolet lamp and observations regarding color were recorded. Using another sterile loop, a loopful of plasmid solution was extracted from the pGlo plasmid DNA stock tube and mixed into the +pGlo tube. However, the plasmid DNA was not added to the –pGlo solution because it is serving as the control group in this experiment. After the solution was mixed completely, both tubes were closed and put on ice for 10 minutes. During the 10 minutes of ice time, the four LB nutrient agar plates were labeled in the following manner: the LB/amp plate was labeled +pGlo, the other was labeled –pGlo, LB/amp/ara was labeled +pGlo, and LB was labeled –pGlo. Ampicillin and arabinose provide for experimental groups to see if the pGlo gene can be shown in the bacteria. After placing the tubes into the microtube rack, both the +pGlo and –pGlo tubes were put into the 42⁰ water bath for 50 seconds then quickly put back on ice for 2 minutes. Next, the rack was removed from the ice and 250 μl of LB nutrient broth was added to both the +pGlo tube and –pGlo tube using a sterile pipette; both tubes were incubated for 10 minutes at room temperature. Once the tubes were removed from the incubator, they were both flicked to mix the solution inside and 100 μl of the transformation and control suspensions was transferred from the tubes to the appropriate nutrient agar plates using a sterile pipette. Using a new sterile loop for each plate, the suspensions were spread gently and evenly around the surfaces of the LB nutrient agar. After stacking the plates and taping them together, they were placed upside down inside of the 37⁰ incubator until the next day. The following day, the agar...
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