Pglo Transformation
Connor Lauffenburger
3/17/13
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
3/17/13
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