Bacterial Transformation Lab
Background: In this lab, you will be performing a
procedure known as bacterial transformation. A
gene is nothing more than a piece of DNA that
codes for a protein. That protein gives the
organism some sort of trait. Genetic
transformation literally involves insertion of a gene
into a new organism in order to change the
organism’s traits. In agriculture, genes coding for
traits such as frost resistance, pest resistance, or
slower spoilage have been genetically added to food crops. 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, where the sick person’s cells are transformed with
healthy copies of the defective gene.
Your task in this lab is to transform bacteria (E. coli) by adding a gene that
codes for Green Fluorescent Protein (GFP). The source of the GFP is the
bioluminescent jellyfish Aequorea victoria. Following transformation, E. coli
will “express” or make the GFP protein and glow a brilliant green color when
placed under ultraviolet light.
In addition to one large circular chromosome, bacteria naturally contain one or
more circular pieces of DNA called plasmids. Plasmid DNA usually contains
genes for one or more traits that may be beneficial to bacterial survival. As an
example, some plasmids contain genes that confer resistance to antibiotics.
Under the right conditions, plasmids are freely exchanged between bacterial
cells.
In this procedure, BioRad has engineered a plasmid that
contains both the gene for GFP and the gene for resistance to
the antibiotic Ampicillin. BioRad has also engineered a
“regulation” system, which allows expression of the new GFP
gene only in the presence of the sugar arabinose—which must be
added to the cell’s nutrient medium. Transformed cells will
therefore appear white on agar plates without arabinose
(because they can’t express the GFP gene), but will appear green under UV light
on agar plates which have had arabinose added.
Please watch the following YouTube video: Bacterial Transformation
PRE-LAB QUESTIONS:
1. What is genetic engineering and bacterial transformation?
2.
What are some ethical considerations involved in genetic engineering?
3.
What are some of the beneficial applications of recombinant DNA technology?
4.
Why is E. Coli HB 101 a good candidate for a bioreactor?
5. What is GFP and where was it originally obtained?
6. In what way(s) can plasmids be an adaptive advantage to bacteria?
7. Why did Bio-Rad encode a gene for ampicillin resistance into the pGLO plasmid?
8. Why is the GFP gene of the pGLO plasmid only “switched on” in the presence of the sugar
arabinose?
9. To genetically transform an entire organism, you must insert the new gene into every cell in the
organism. Which organism is better suited for total genetic transformation – one composed of
many cells, or one composed of a single cell?
10. Scientists often want to know if the genetically transformed organism can pass its new traits on
to its offspring and future generations. To get this information, which would be a better
candidate for your investigation: an organism in which each new generation develops and
reproduces quickly, or one which does this more slowly?
11. Safety is another important consideration in choosing an experimental organism. What traits or
characteristics should the organism have (or not have) to be sure it will not harm you or the
environment?
12. Based on the above considerations, which would be the best choice for a genetic
transformation: a bacterium, earthworm, fish or mouse? Describe your reasoning.
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Figure 1:
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A Bacterial Cell
Label the 4 Parts of the cell indicated by the lines pointing to structures.
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