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Class notes biol 2281

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Course
Biol 2281

Institution
University Of Texas - Dallas

In today’s biology lab, we investigated the transfer of bacteria using different methods of inoculation. We began by preparing agar plates with nutrient media and then performed streak plating to isolate colonies from a sample. Each group introduced bacteria using sterile swabs, ensuring proper a...

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Biol 2281, Fall 2024
E8: Bacterial Transformation Procedure/Report

Experiment 8: Bacterial Transformation

Objectives:
At the end of this exercise, you will be able to
1. Understand the concepts of plasmids and genetic transformation.
2. Perform a bacterial transformation with appropriate laboratory techniques.
3. Describe the function of a selectable marker in the isolation of transformants.
4. Analyze the results and calculate the transformation efficiency.

This topic was adapted from pGLOTM Bacterial Transformation kit, Bio-Rad Laboratories, Inc. Duplication of
any part of the document is permitted for classroom use only.

One of the most important techniques of modern molecular biology is the ability to introduce a specific
gene into an organism and have that genetic information expressed by the organism. This process,
called genetic transformation, played a critical role in the history of molecular biology and the
discovery that DNA was the genetic material. Four scientists, Griffith in the 1920's and Avery,
McCarty, and MacLeod in the 1940's, showed that the apparent "transformation" of one bacterial type
into another required DNA and no other biological molecule.

Today, transformation is not limited to bacteria, and in fact, is used routinely by molecular biologists
to manipulate and study the behavior of genes. 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 (Escherichia coli K-12:HB101) 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. 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 (p=plasmid) encodes the gene for GFP and a gene (bla) for beta-
lactamase that provides 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 (see Appendix ). Selection for cells that have been transformed with pGLO
DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type

1

, Biol 2281, Fall 2024
E8: Bacterial Transformation Procedure/Report
phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the
nutrient agar medium. Arabinose (C5H10O5) is found in nature and can be metabolized by
microorganisms as a carbon source.

In the lab, E. coli cells are more likely to incorporate foreign DNA if their cell walls and cell
membrane are altered so that DNA can pass through more easily. Such cells are said to be
"competent". E. coli cells are made competent by a process that uses calcium chloride and heat shock.
In addition, cells from exponential phase of growth are typically used in preparation of competent cells
since they give better transformation efficiency.

The transformation procedure involves three main steps. These steps are intended to introduce the
plasmid DNA into the host cells and provide an environment for the cells to express their newly
acquired genes.
 Incubation: the cells mixed with a transformation solution of CaCl2 (calcium chloride) and the
plasmid.
 Heatshock: the cells and DNA mixture are then subjected to a treatment at 42ºC for 50
seconds; the temperature changes destabilize cell membranes and create thermal imbalance.
 Recovery: a short incubation period to begin expressing their newly acquired genes

Appendix

MAP of pGLO

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