Transformation Of Escherichia Coli With pGLO Plasmid
April 24, 2013
This experiment focuses on genetic engineering and transformation of bacteria. The characteristics of bacteria are altered from an external source to allow them to express a new trait, in this case antibiotic resistance. In is experiment foreign DNA is inserted into Escherichia coli in order to alter its phenotype. The goal of the experiment is to transform E. coli with pGLO plasmid, which carries a gene for ampicillin resistance, and determine the transformation efficiency. The bacteria are transformed by a combination of calcium chloride and heat shock. When the bacteria are incubated on ice, the fluid cell membrane is slowed and then the heat shock increases permeability of the membrane. The results obtained in the experiment show that the E. coli that was transformed with pGLO was able to resist ampicillin and grow in its presence. These results suggest that microorganisms can be genetically engineered to selectively resist certain contaminants, which means that they can potentially be used to human benefit to rid the environment, or even the human body, of unwanted toxins.
Transformation occurs when altered genetic characteristics of bacteria are acquired from a different source. Plasmids are used to transform bacteria because they are small pieces of DNA capable of independently replicating and therefore transferring their (often beneficial) traits to the bacteria. The goal of genetic transformation in this experiment is for the bacteria Escherichia coli to obtain an antibiotic resistance to ampicillin, which can be physically observed when the bacteria expresses the reporter gene Green Fluorescent Protein (GFP) because the transformed bacteria will glow green under UV light when in the presence of arabinose. The gene for GFP is naturally found in a bioluminescent jellyfish, allowing it to glow in the dark. The plasmid used to transform the bacteria contains the antibiotic resistance along with a gene that codes for the fluorescent protein.
The objective of this experiment is to genetically engineer ampicillin resistant E. coli by transforming it with a plasmid containing an antibiotic resistance gene and a gene that codes for GFP and to calculate the transformation efficiency. This will alter the phenotype by inserting foreign DNA. This experiment is important because it demonstrates a technique that has practical applications in areas such as environmental concern. Bacteria that have been genetically altered can obtain traits that allow it to be used to break down toxic compounds (i.e. oil spill) to reduce the amount of harmful substances found in soil and water (Spilios, 2013). Some environmental contaminants are actually toxic for microorganisms, like bacteria, and deactivate the cells abolishing their ability to break down the contaminants (Pieper, 2000). For this reason, bacteria that are resistant to specific toxins are preferable and it would be desirable to replicate their DNA– the resistance gene specifically –and use it to create such resistance in other organisms. This research builds directly from the knowledge used in this experiment transforming E. coli.
The hypotheses for this experiment are that the bacteria will be transformed by the plasmid to develop an antibiotic resistance and will grow despite the presence of ampicillin. Additionally, when arabinose sugar is added to the strain of bacteria transformed by the plasmid, it will express GFP and glow green under UV light.
The methods and procedures for transforming bacteria in this experiment were performed according to Spilios (2013). Bacteria strains of E. coli were genetically transformed with a plasmid to carry ampicillin resistance. 250 microliters of a CaCl2 transformation solution were pipetted into closed test tubes, which were then placed on ice. A starter plate with E. coli colonies was observed and two...
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