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Lab 7 Genetic Transformation

Lab 7 Genetic Transformation - Biology 05LA Winter Quarter...

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Biology 05LA – Winter Quarter 2010 Lab 7 – Page 1 LAB 7: GENETIC TRANSFORMATION* Genetic transformation is the act of altering the genotype of a cell by the introduction of DNA molecules from an external source. In nature, the source of DNA can simply be a virus or a DNA molecule that was released into the environment when a cell died and its DNA exposed. You probably have already heard about genetic transformation in lecture (i.e., Griffith's experiments involving the transformation of rough to smooth bacteria as part of his discovery that nucleic acids were in fact the genetic material). Today genetic transformation is used in the laboratory to engineer prokaryotes, animals, and plants for enhanced phenotypic characters and to repair deficiencies. The transformation process itself involves two distinct steps: 1. the entry of foreign DNA into the cell and 2. the expression of the genes residing on the foreign DNA molecule. In this experiment, you will be provided with bacterial plasmid DNA as a source of transforming DNA. Plasmids are very small, circular molecules that occur in nature. Plasmids replicate independently inside the bacterial cell. They have been manipulated by scientists for movement of genes in and out of bacteria in the laboratory. The plasmid we are using in today's experiment is called pUC118. It contains a gene that confers resistance to the antibiotic ampicillin. Bacteria that contain the plasmid can remain alive and grow in the presence of this antibiotic. Bacteria without the plasmid stop growing and may die in the presence of this drug. (Note: ampicillin is a derivative of penicillin). The objective of today's experiment is to genetically transform ampicillin-sensitive (or susceptible) bacteria into antibiotic-resistant bacteria. The addition of a plasmid with an ampicillin-resistance gene will thus change the genotype and the phenotype of the bacterial cell. Unlike the Stretptococcus (formerly Pneumococcus ) studied by Griffith, Escherichia coli cells do not spontaneously take up DNA. Therefore prior to this laboratory, E. coli cells were treated with CaCl 2 to enhance DNA uptake; these cells are “competent” for DNA uptake. Using the procedure described below, each group of students will mix DNA with the competent E. coli cells. Any cell that takes up the plasmid DNA and maintains it to express ampicillin-resistance is called a transformant. One third of the cells will be plated on ampicillin plates to determine the number of transformants. One third of the cells will be plated on l uria b roth agar (LB) plates as controls. The third portion of cells will be serially diluted and plated on antibiotic-free media. The latter will be used to determine the total number of bacterial cells that were available for transformation. After growth, each lab group will score the number of transformants obtained and determine the total number cells available for transformation. These figures will be used to calculate transformation efficiency which, for our
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