pGLO™ Bacterial Transformation

Introduction

Introduction to Transformation

In this lab, you will perform a procedure known as genetic transformation. Genetic transformation literally means “change caused by genes”, and occurs when the cell incorporates and expresses a new piece of genetic material – DNA derived from another organism. Transformation involves the insertion of a gene into an organism in order to alter the recipient organism’s expression. Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest and breakdown pollutants such as oil spills or heavy metals contamination In medicine, disorders caused by defective genes are being treated by gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of the defective gene.

Your Protein of Interest - The Green Fluorescent Protein, GFP

You will use a procedure to transform bacteria with a gene that codes for Green Fluorescent Protein, GFP. The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria. Following the transformation procedure, the bacteria will express their newly acquired gene and produce GFP, which causes them to glow a brilliant green color under ultraviolet or blue light.

The Host Cell - Escherichia coli (E. coli)

The bacterium, E. coli, is the ideal host for transformation because it is a small, single-celled organism that reproduces quickly, so its transformation will be seen rather quickly. Also, the strain of E. coli being used is nonpathogenic, does not make people or animals sick, and it does not survive outside the laboratory environment. Although it is safe, it requires the use of Standard Microbiological Practices, as directed by your instructor.

Objectives

In this experiment, you will

• Use a plasmid vector to transform bacteria with genes for Green Fluorescent Protein (GFP) and antibiotic resistance in a controlled experiment.
• Use the heat shock method of transforming E. coli.
• Regulate the expression of the GFP gene using arabinose.
• Describe the biological process involved in transforming bacterial cells.
• Calculate your transformation efficiency.
• Learn basic molecular biology techniques.