BME 100L Fall 2009 INDUCIBLE GENE CIRCUIT Safety: The Escherichia colibacteria used in this lab have been modified to be non-virulent, but may induce an acute immune response in a very small percentage of individuals (this may occur whenever one is exposed to foreign materials or organisms). You must take the following safety precautions: •Wash your hands when entering and before exiting the lab •Wear a lab coat and gloves (dispose it after the lab) •No eating or drinking in the lab •Wear closed toe shoes Objectives: In this lab, you will: •Week 1 – isolate plasmid DNA containing a reporter gene; transform plasmid into E. colicells •Week 2 – analyze cells from the previous week Background: Plasmids and Transformation A plasmid is a circular piece of double-stranded DNA. Plasmids live in bacterial cells naturally, usually providing an advantageous gene for the bacteria (if the plasmid does not provide some advantage for the bacteria, it will be lost from the bacterial cells). Researchers utilize this relationship by “transforming” plasmids carrying desired DNA sequences into bacterial cells. “Transformation” simply means that plasmid DNA is taken up by bacterial cells. Bacterial cells that have been treated so that they can easily take up DNA are called “competent cells.” Once transformed into a bacterial cell, the plasmid DNA remains separate from the cell’s genomic DNA. Using the cellular machinery, the plasmid used in this lab can replicate independently; thus, the plasmid replicates more often than the bacterial cell divides. This results in many copies of the plasmid existing in a single bacteria cell. In this way, the bacterial culture acts as a factory to produce a large quantity of plasmid DNA. Steps must be taken to ensure that the plasmid is maintained in the cells. Plasmids are engineered to contain genes for antibiotic resistance. This allows researchers to selectively grow bacteria that contain the plasmid (and thus, the desired DNA sequences) by using growth media with the appropriate antibiotic. The plasmid used in this lab carries a gene that makes transformed cells resistance to the antibiotic kanamycin. Thus, growth media with kanamycin will be used. Only bacterial cells that contain the plasmid will grow in media containing the antibiotic. Each individual colony that grows on the LB + kanamycin plate is derived from a single transformed bacterial cell.
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