pGLO Transformation
Introduction to Transformation
In this lab you will perform a procedure known as genetic transformation. Remember that a gene is
a piece of DNA which provides the instructions for making (codes for) a protein. This protein gives an
organism a particular trait. Genetic transformation literally means “change caused by genes,” and
involves the insertion of a gene into an organism in order to change the organism’s trait. 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 oil spills. In medicine,
diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically
transforming a sick person’s cells with healthy copies of the defective gene that causes the disease.
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
. Green
Fluorescent Protein causes the jellyfish to fluoresce and glow in the dark. Following the transformation
procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein,
which causes them to glow a brilliant green color under ultraviolet light or the correct wavelength of blue
light.
In this activity, you will learn about the process of moving genes from one organism to another with
the aid of a plasmid. In addition to one large chromosome, bacteria naturally contain one or more small
circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that
may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing
them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new
environments. The recent occurrence of bacterial resistance to antibiotics is due to the transmission of
plasmids.
Bio-Rad’s unique pGLO plasmid encodes the gene for GFP and a gene for resistance to the antibiotic
ampicillin. pGLO also incorporates a special gene regulation system, which can be used to control
expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in
transformed cells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have
been transformed with pGLO DNA is accomplished by growth on ampillicin plates. Transformed cells will
appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green under blue
light when arabinose is included in the nutrient agar medium.
You will be provided with the tools and a protocol for performing genetic transformation.
Your task will be to:
1. Do the genetic transformation.
2. Determine the degree of success in your efforts to genetically alter an organism.
Consideration 1: How Can I Tell if Cells Have Been Genetically Transformed?
Recall that the goal of genetic transformation is to change an organism’s traits, also known as
their phenotype. Before any change in the phenotype of an organism can be detected, a thorough
examination of its natural (pre-transformation) phenotype must be made. Look at the colonies of
E.
coli
on your starter plates. List all observable
traits or characteristics that can be described and answer the following questions in your lab
notebooks:
