Vibrio Fischeri Luciferase Lab Report

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To study the expression of the Vibrio fischeri luciferase operon in Escherichia coli (E. coli) by isolating and purifying a plasmid from E. coli, determining its orientation with respect to the plasmid backbone by restriction mapping, and transforming it into an alternate E. coli strain.

Each individual received either E. coli A or B strain of pKN800 plasmid DNA. The plasmid was purified and isolated, then cut with PstI restriction enzyme to create a sample of PstI-cut and uncut pKN800 plasmid DNA and to distinguish orientation A from B. These samples were loaded onto a 0.8% agarose gel for electrophoresis. Both cut and uncut plasmid DNA were transformed into E. coli DH5α. Finally, the transformed DNA was plated on LB and LB with ampicillin agar plates.

The procedure for this experiment was followed on pg. 16-23 of the MB 311 laboratory manual. One exception to the protocol was step D-12 on pg. 22: All plates were incubated for three days at 300C.

To determine the orientation
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This restriction enzyme cut the plasmid into two fragments as shown in Figure 1b. The sizes of the fragments on the gel indicate what orientation the plasmids are in. For example, the pKN800A-cut lane had two fragments of 10,500 bp and 1,500 bp while pKN800B-cut lane had fragment sizes of approximately 10,000 bp and 3,500 bp. The intensity of the fragments on the gel has a positive correlation with the size of the plasmid; the band intensity becomes more faint as the fragment sizes decreases. The distance the plasmid DNA migrates is dependent on whether the DNA is completely supercoiled or contains a break in one strand. Circular DNA with a break tends to migrate through agarose much more slowly than supercoiled circular DNA of the same size. Based on this information, the orientation of the luciferase operon of pKN800A is in the same direction as the tet gene, or

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