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Transformation of Escherichia coli Using an Inducible Expression Vector Containing the Bioluminescent Vibrio fischeri Lux Operon by Bryan Hart & Crystal Harmon Bioluminescence biologically mediated synthesis of compounds that react to emit visible light energy found in diverse range of species fungi, insects, algae, free living bacteria, mollusks, crustaceans, and other animals in symbiosis with bioluminescent bacteria Evolutionarily speaking based upon reproductive communication and competition attract mates or advertise high fitness levels (remember energy allocation from EvoEco?) illumination for predation or protection ex. fireflies, cuttlefish, dragonfish or just to look cool Dragonfish Comb Jelly Firefly Panellus stypiticus Vibrio fischeri common bioluminescent bacteria in photophores (light organs) of marine organisms Gram negative, f. Vibrionaceae • pathogenic and symbiotic interactions with animal tissue • virulent pathogens of crustaceans, also free living saprophytic cells in seawater • symbiosis established by inoculation of juvenile animal hosts V. fischeri streak plate the Lux operon gene group responsible for bioluminescence, synthesizes luciferase, key catalyst consists of 8 main genes three parts: regulatory genes, fatty acid reductase polypeptides, and luciferase subunits luxR luxI luxCDABEG Luciferase Cycle Protocol in a nutshell extract Vibrio fischeri DNA w/ DNeasy® Tissue Kit create genomic library w shotgun cloning • Sal I restriction digest of the chromosome • ligate restriction fragments into inducible Promega pGEM® -3Zf(+) vector • transform BL21 (DE3) E. coli w/ cloned vectors • select correctly transformed colonies by blue-white screening (and possibly bioluminescence) manipulate lux expression in successfully transformed cells Why Sal I? cleaves a six base pair palindromal sequence (GTCGAT) w/ sticky ends restriction fragment length of 4000 bp from average genome, but this may vary due to G+C content but most importantly… the lux operon exists on a Sal I restriction fragment of around 9kb Why pGEM® -3Zf(+) ? T7 Sal I lacZ Amp Why BL21 (DE3) E. coli ? laboratory strain with the gene encoding T7 RNA polymerase conveniently under lac operon control induce/repress with carbs or analogs expression of lux operon through direction of lac operon- E. coli media compatible Shine-Dalgarno sequences Timeline Week of Sept 13th – 15 pts Week of Sept 20th – 15pts Week of Sept 27th – 10pts Week of Oct 3rd – 5pts Week of Oct 10th – 5pts Until Nov 22nd- receive vector plasmid and DNeasy , begin DNA extraction chromosomal and vector digestion, gel verification ligation and gel verification prepare competent cells, transformation, and selection manipulation of operon possibly redoing steps… Budget Promega pGEM® -3Zf(+) vector $66.00 DNeasy Tissue Kit (50) $110.00 T4 DNA ligase $33.00 Sal I $55.00 Total $264.00 References Altman, John. Autoinduction of Expression in the T7 Expression System. Altman Laboratory at Emory Vaccine Center. 3 Sept. 2004. http://www.microbiology.emory.edu/altman/f_protocols/f_tetramers/ autoind_annot.html Bluth, Brian J., Sarah E. Frew, and Brian McNally. Cell-Cell Communication and the lux operon in Vibrio fischeri. Carnegie Mellon University. 3 Sept. 2004. http://www.bio.cmu.edu/courses/03441/TermPapers/97TermPapers/ lux/default.html Promega Bacterial Expression Vectors. Promega Corporation. 3 Sept. 2004. http://www.promega.com/vectors/bacterial_express_vectors.html Slock, James. Molecular Biology Experiments Utilizing the lux Genes of Vibrio fischeri and gfp Gene of Aequoria victoria. King’s College PA. 3 Sept. 2004. <http://www.kings.edu/biology/lux/luxbiolum.html> Winfrey, Michael, Marc Rott, and Alan Wortman. Unraveling DNA Molecular Biology for the Laboratory. New Jersey: Prentice Hall, 1997.