6Thurs_Transformation

6Thurs_Transformation - Lab Exercise: Transformation...

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OBJECTIVES 1. Understand the process of transformation and how it is used in a laboratory setting for expression of genes (i.e. production of proteins). 2. Perform a successful transformation using the pTOM plasmid. INTRODUCTION Genetic transformation is used in many areas of biotechnology, and, at its heart, requires two things: Donor DNA and recipient cells. Cells which receive the donor DNA are considered genetically recombined , that is, they have their original DNA (their chromosome) and new DNA (the plasmid) and whatever genes that plasmid carries. Before considering the details of recombination, we will consider each of these players individually. Plasmids were discovered as extra-chromosomal genetic material in the late 1960s. Like the bacterial chromosome, they are circular but they are much smaller (2,000–10,000 bp), and usually contain 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 and adapt to new environments. For instance, the quick dissemination of bacterial resistance to antibiotics is due in part to the transmission of plasmids. These naturally occurring plasmids have been engineered to contain not only antibiotic re s i s t an ce (wh i i s sed in the l abo r atory as a selective marker for successful transformation) but other “ genes of interest .” If a plasmid is transformed into an E. coli cell that is sensitive to the antibiotic ampicillin, it will confer resistance to that antibiotic. Grow ing the transformants in the presence of amp ic i l l in is an easy way to se lect for recombined E. coli cells. However, E. coli that have become antibiotic-resistant are not themselves useful in biotechnological applications. As such, plasmids must be engineered to include some other gene of interest. This gene of interest is spliced into the plasmid at its multiple cloning site. Many plasmids are commercially available, and many contain the following components: 1. A selectable marker ( i.e ., a gene that encodes for antibiotic resistance);
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This note was uploaded on 12/23/2009 for the course BIO 205 taught by Professor Murphy during the Fall '09 term at Miramar College.

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6Thurs_Transformation - Lab Exercise: Transformation...

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