Experiment: Creating a Genomic Library of Vibrio DNA and Cloning the Lux Operon
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Detecting glowing E.Coli Colonies by making recombinant DNA from the lux operon of Vibrio Fischeri to pGEM.
Liao, Tffany The marine bacterium Vibrio Fischeri produced bioluminescence effect due to lux operon transcription. The purpose of the experiment is to create a genomic library of Vibrio DNA and clone the lux operon by making Recombinant DNA and transform into another organism, E. Coli. Chromosomal DNA of vibrio fischeri was first extracted and digested with restriction enzyme Sal I, then ligated with the vectors and transformed into the E. Coli cells. A few white colonies indicating the E. Coli cells took up the hybrid plasmids were observed on the plate but no glowing colonies were detected. The lux operon was not successfully cloned in this experiment.
Introduction Vibrio Fischeri possesses a system called quorum sensing as a mean to express bioluminescence and communicate collectively with other bacteria. (Khajanchi, 2011). Quorum sensing involves using signaling molecules called autoinducers transcribed from Lux I of lux operon for bacteria to coordinate their behavior depending on the environment (Aheml, 2004; Kaplan, 1985). If the bacterial population density increases in the environment, autoinducers will accumulate and combines with the gene products of lux operon to produce bioluminescence. (Rutherford, 2012) Lux Operon contains seven Lux genes in specific orders. Lux CDABE are located downstream of the promoter and LuxR, which is transcribed in the opposite direction as all other genes, is located upstream of the promoter (Swartzman, 1990). When lux I-transcribed autoinducers accumulate and achieve a critical concentration in the environment, they will bind to the product of lux R and initiate the transcription and translation and produce luminescence effects. (Engerbrecht, 1983). Lux A and lux B are genes in the lux operon to transcribe the enzyme luciferase that is essential to catalyze the bioluminescence reaction with fatty acid
Detecting glowing E.Coli Colonies by making recombinant DNA from the lux operon of Vibrio Fischeri to pGEM.
Liao, Tffany The marine bacterium Vibrio Fischeri produced bioluminescence effect due to lux operon transcription. The purpose of the experiment is to create a genomic library of Vibrio DNA and clone the lux operon by making Recombinant DNA and transform into another organism, E. Coli. Chromosomal DNA of vibrio fischeri was first extracted and digested with restriction enzyme Sal I, then ligated with the vectors and transformed into the E. Coli cells. A few white colonies indicating the E. Coli cells took up the hybrid plasmids were observed on the plate but no glowing colonies were detected. The lux operon was not successfully cloned in this experiment.
Introduction Vibrio Fischeri possesses a system called quorum sensing as a mean to express bioluminescence and communicate collectively with other bacteria. (Khajanchi, 2011). Quorum sensing involves using signaling molecules called autoinducers transcribed from Lux I of lux operon for bacteria to coordinate their behavior depending on the environment (Aheml, 2004; Kaplan, 1985). If the bacterial population density increases in the environment, autoinducers will accumulate and combines with the gene products of lux operon to produce bioluminescence. (Rutherford, 2012) Lux Operon contains seven Lux genes in specific orders. Lux CDABE are located downstream of the promoter and LuxR, which is transcribed in the opposite direction as all other genes, is located upstream of the promoter (Swartzman, 1990). When lux I-transcribed autoinducers accumulate and achieve a critical concentration in the environment, they will bind to the product of lux R and initiate the transcription and translation and produce luminescence effects. (Engerbrecht, 1983). Lux A and lux B are genes in the lux operon to transcribe the enzyme luciferase that is essential to catalyze the bioluminescence reaction with fatty acid