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The emission of light by living organisms is a fascinating process. The genetic system required for luminescence in the bacterium Photobacterium (Vibiro) fischeri is the lux operon. This operon contains a gene for luciferase (the enzyme that catalyzes the light-emitting reaction) and genes for enzymes which produce the luciferins (which are the substrates for the light-emitting reaction.). In this exercise, students create a luminescent population of bacteria by introducing into E.coli a plasmid that contains this lux operon. The success of the transformation is readily apparent since the E.coli colonies that take up this plasmid glow in the dark as shown below. The simple procedure can be carried out during a single 1-1.5 hour laboratory session. Sufficient sterile materials are provided for sixteen platings and the experiment is designed for 8 groups of students. Additional experiments can be performed following completion of this experiment including: 1. Analysis of the effects of growth temperature and time on the glowing process. This analysis can be carried out by replating the transformed cells onto fresh nutrient agar-ampicillin plates, which can be purchased separately (Click Here) 2. Isolation of plasmid lux from transformed cells using the plasmid DNA isolation kit (cat. # 3-18) that is described by Clicking Here.
PROCEDURE AND RESULTS 1. Add E.coli to calcium chloride solution. 2. After 15 minutes, add control plasmid and plasmid lux to two tubes of cells and incubate both on ice for 10 minutes. 3. Incubate the tubes at 37°C, add nutrient broth and then spread the bacterial suspensions onto agar plates. 4. View the plates in the light and in the dark after two days at room temperature. Control Plasmid Plasmid Lux
Light
Dark
The photograph in the bottom panel was taken in total darkness using only the light emitted from the bacteria that were transformed with plasmid lux.
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