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 reductase complex transcribed from lux C, D and E. (Meighen, 1988). The overall purpose of this experiment is to create a genomic library and clone the luxAB genes from the marine bacterium Vibrio Fischeri by using Recombinant DNA. Restriction enzymes are used to digest the genomic DNA and create DNA fragments as inserts. The inserts are then combined with the vectors and ligated by ligases in order to transform the recombinant
DNA into the host. The ultimate goal is to express the lux operon gene products to produce bioluminescence in another organism, E. Coli that is not capable of expressing the light naturally. The article “High-efficiency transformation of mammalian cells by plasmid DNA” uses a similar approach to clone a specific gene. (Chen, 1987) We hypothesize that if the hybrid plasmids containing lux operon is successfully transformed into the E. Coli DH5-alpha cells, the cells will produce bioluminescence and glow in the dark. The biggest implications that will impact the success of this experiment is the possibility that we will lose the gene of interest during the process. Therefore it is important to use gel electrophoresis to check if digestion and ligation work to ensure the gene of interest is being cut and inserted properly. Material and Method Part I: Isolation of genomic DNA of vibrio fischeri. The culture of V. fischeri is first centrifuged to separate the cells from supernatant. The following reagents are added to destabilize the outer membrane of the cells and denature the proteins: TES buffer, lysozyme, proteinase K and SDS. Phenol is then introduced to separate the proteins and lipids from the nucleic acids. Ethanol is added after removing the aqueous phase of DNA to aggregate DNA structures. Use a hook to wind the DNA and turn it upside down to drain and get rid of all the ethanol. Place the DNA into TE buffer again and add RNAse and proteinase K to discard RNAs and further purify DNA. Phenol and chloroform are introduced to further...
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