Lab 9: Bacterial Transformation with pGLO
ο Practice formulating hypotheses, predictions, and experimental design. ο Describe the principles of bacterial transformation.
ο Explain the procedure for gene transfer using plasmid vectors. ο Induce the transfer of the pGLO gene (in a plasmid) into E. coli. ο Describe the traits carried by the pGLO gene.
ο Describe how to activate (“turn on”) the pGLO gene.
ο Describe how to recognize the transformed cells (from this lab). ο Know the terms used in this lab including transformation (in this case transformation does NOT mean the conversion of a normal cell to a cancerous one), vector, plasmid, fluorescence, antibiotic resistance, E. coli. ο Answer the questions posed in this lab.
ο Read about the control of gene expression on pages 353-356 and about transformation on page 348 of the textbook.
ο Read this lab and be ready to begin the exercises.
ο Define the following terms (but do not hand in): transformation, vector, plasmid, fluorescence, antibiotic resistance, E. coli
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 creates 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. 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.
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