lab_15_pBluescript_DNA_transformation - Miramar College...

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Lab Exercise 15: pBluescript Transformation: Blue/White Colony Selection Page 1 of 5 Miramar College Biology 205 Microbiology Lab Exercise 15: pBluescript Transformation: Blue/White Colony Selection Background The natural phenomenon of horizontal gene transfer is exploited in the biotechnology lab to produce recombinant bacterial cells. One such transfer of genetic transformation is transformation . Genetic transformation 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. Because this plasmid carries with it foreign DNA ( e.g . the gene to produce human growth hormone), it is called a vector ( i.e. a delivery mechanism) for that DNA. 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 transfer plasmids back and forth, allowing them to share these beneficial genes and survive in otherwise hostile environments. These naturally occurring plasmids have been engineered to contain not only antibiotic resistance (which is used in the laboratory 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. Growing the transformants in the presence of ampicillin is an easy way to select 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. For this to happen, a plasmid containing resistance to an antibiotic ( e.g ., ampicillin) is used as a vector . The gene of interest is inserted into the vector plasmid at its multiple cloning site - an area of the plasmid that contains many restriction enzyme recognition sites within an inducible reporter gene. This recombinant plasmid is then put into ampicillin - sensitive E. coli . When the bacteria are grown in the presence of ampicillin, their plasmid-mediated ampicillin resistance allows only those cells which are recombinant to survive. As long as the cells are grown in media containing ampicillin, they will need the plasmid to survive and will continue to replicate it, along with your gene of interest. Because each will exhibit unique phenotypes, they are easily distinguished from one other. Many plasmids are commercially available, and all of them contain four key components:
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lab_15_pBluescript_DNA_transformation - Miramar College...

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