Download 08 Bacterial Transformation Lab Part1 Fa08

Lab #8: Bacterial Transformation
OBJECTIVES:
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Practice formulating hypotheses, predictions, and experimental design.
Describe the principles of bacterial transformation.
Explain the procedure for gene transfer using plasmid vectors.
Induce the transfer of the pGLO gene (in a plasmid) into E. coli.
Describe the traits carried by the pGLO gene.
Describe how to activate (“turn on”) the pGLO gene.
Describe how to recognize the transformed cells (from this lab).
General Procedures
 You will work in groups of 2 or 4 (as specified by the instructor), but all students will complete
their own lab questions/data sheet & turn it in individually.
 Read the lab exercise and follow the directions carefully. In the second lab period you will
analyze your results.
INTRODUCTION
In this lab you will perform a procedure known as a genetic transformation. Remember that
a gene is a piece of DNA that provides the instructions for making (coding for) a protein that gives an
organism a particular trait. Genetic transformation literally means change caused by genes and it
involves the insertion of a gene(s) into an organism in order to change the organism's trait(s). 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 bio-remediation,
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 gene involved in their disease.
You will use a procedure to transform bacteria with a gene that
codes for a Green Fluorescent Protein (GFP). The real-life source of
this gene is the bioluminescent jellyfish Aequorea victoria. The gene
codes for a Green Fluorescent Protein that 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 that causes them to glow a brilliant
green color under ultraviolet 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, which create the
opportunity for them to share these beneficial genes. (Note that the bacteria don’t know that they are
picking up 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.
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The unique plasmid we use encodes the gene for the Green Fluorescent Protein (GFP) and a
gene for resistance to the antibiotic, ampicillin. The plasmid also incorporates a special gene
regulation system, which can be used to control expression of the fluorescent protein in transformed
cells. The gene for the Green Fluorescent Protein can be switched on in transformed cells by adding
the sugar, arabinose (ara), to the cells’ nutrient medium. Selection for cells that have been
transformed with the plasmid DNA is accomplished by growth on antibiotic plates. Transformed
cells will appear white (wild type phenotype) on plates not containing arabinose, and fluorescent
green under UV light when arabinose is included in the nutrient agar.
You will be provided with the tools and a protocol for performing genetic transformation in
Escherichia coli. This transformation procedure involves three main steps. These steps are intended
to introduce the plasmid DNA into the E. coli cells and provide an environment for the cells to
express their newly acquired genes. Many species of bacteria have special membrane proteins for the
uptake of DNA from the external environment. E. coli does not appear to have these types of
membrane proteins; however, placing E. coli in a relatively high concentration of calcium ions and
performing a procedure called “heat shock” will stimulate these cells to take up pieces of foreign
DNA.
To move the plasmid DNA through the cell membrane you will:
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Use a transformation solution of CaCl2 (calcium chloride)
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Carry out a procedure referred to as heat shock
For transformed cells to grow in the presence of ampicillin you must:
1. Provide them with nutrients and a short incubation period to begin expressing their newly
acquired genes
PROCEDURES
Part 1: Bacterial Transformation
Exercise A: Introduction to Sterile Technique (in lab session)
You will practice using sterile technique, as instructed at the beginning of lab session, before
you do the experiment. When culturing bacteria, you must not introduce other, contaminating
bacteria into your culture. Potentially contaminating bacteria are ubiquitous; they are found
everywhere (including on the bench top and on your hands). It is especially important to keep the
inoculation loops, the pipette tips sterile, and the surfaces of the agar plates must not touch or be
touched by any contaminating surface.
Exercise B:
Bacterial Transformation (in lab session)
MATERIALS & PROCEDURES
1. Follow the procedures in the “Transformation Kit-Quick Guide” provided in lab.
2. The plates will be incubated for 24-48 hours, and then placed in a refrigerator to slow the growth
of the bacteria. You will than observe the plates in the next lab period to collect your data.
3. Complete your lab report:
 formulate a hypothesis on which this investigation is based, of how E. coli cells can be
transformed by the pGLO plasmid,
 formulate the predictions, and
 explain the experimental design.
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