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GENETIC TRANSFORMATION OF BACTERIA WITH THE GENE FOR GREEN FLUORESCENT PROTEIN (GFP) LAB BAC3 Adapted from "Biotechnology Explorer pGLO Bacterial Transformation Kit Instruction Manual". (Catalog No. 166-0003-EDU) BIO-RAD Laboratories, 2000 Alfred Nobel Drive, Hercules CA 94547. STANDARDS ADDRESSED 3.1.10A/3.1.12A Apply concepts of systems and control at the molecular level to assess the outcome of bacterial transformation. 3.2.10B/3.2.12B Describe the transformation results using precise quantitative and qualitative skills based on observations. Evaluate the experimental data correctly within experimental limits. 3.3.10C/3.3.12C Describe how genetic information is inherited and expressed at t the molecular level. KEY CONCEPTS: Genetic transformation, plasmid DNA, cloning, restriction enzymes, antibiotic selection, gene regulation, transcription, protein expression INTRODUCTION Genetic transformation is the process by which a gene or genes from one organism are transferred to another organism via an engineered molecule of DNA. If the procedure is successful, the organism is capable of producing the protein encoded by the transformed gene, thus creating a genetic change. Genetic transformation is commonly used in biotechnology. In agriculture, transformation is used to confer genes for pest, frost and spoilage resistance. Transformation of the human insulin gene into bacteria has allowed for production of the protein on a large scale. 1 To aid in bioremediation of oil spills, bacteria are transformed with genes that allow them to digest toxic components of the oil. 2 The procedure contained in this lab will allow for the transformation of bacteria with a gene from the bioluminescent jellyfish, Aequorea victoria . A successful transformation will result in the expression of the green fluorescent protein (GFP) in the bacteria, causing them to glow bright green under long-wave UV light. 1 http://www.littletree.com.au/dna.htm 2 http://www.epa.gov/ORD/NRMRL/lrpcd/esm/oil_spill_bioremediation_researc.htm _______________________________________________________________________ Westminster College SIM MICRO3-1
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Transformation of Bacteria with GFP Transformation and Antibiotic Selection : Genetic transformation in this laboratory will be facilitated by using the pGLO plasmid (see below). A plasmid is a circular, self- replicating DNA molecule which can be contained in a bacterial host cell without interfering with the function of the bacterial chromosome. Bacteria are capable, on their own, of randomly acquiring small pieces of DNA from their environment, but the process is inefficient. The transformation protocol in this lab uses a chemical, calcium chloride (CaCl 2 ), plus heat to increase the efficiency of DNA uptake by the bacterial cell. Even with the chemical transformation procedure, not
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