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Transformation of Escherichia coli Using an Inducible Expression Vector Containing the Bioluminescent Vibrio fischeri Lux Operon by Bryan Hart & Crystal.

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Presentation on theme: "Transformation of Escherichia coli Using an Inducible Expression Vector Containing the Bioluminescent Vibrio fischeri Lux Operon by Bryan Hart & Crystal."— Presentation transcript:

1 Transformation of Escherichia coli Using an Inducible Expression Vector Containing the Bioluminescent Vibrio fischeri Lux Operon by Bryan Hart & Crystal Harmon

2 Bioluminescence biologically mediated synthesis of compounds that react to emit visible light energy biologically mediated synthesis of compounds that react to emit visible light energy found in diverse range of species found in diverse range of species fungi, insects, algae, free living bacteria, mollusks, crustaceans, and other animals in symbiosis with bioluminescent bacteria fungi, insects, algae, free living bacteria, mollusks, crustaceans, and other animals in symbiosis with bioluminescent bacteria

3 Evolutionarily speaking based upon reproductive communication and competition based upon reproductive communication and competition attract mates or advertise high fitness levels (remember energy allocation from EvoEco?) attract mates or advertise high fitness levels (remember energy allocation from EvoEco?) illumination for predation or protection illumination for predation or protection ex. fireflies, cuttlefish, dragonfish ex. fireflies, cuttlefish, dragonfish or just to look cool or just to look cool

4 Dragonfish Comb Jelly Panellus stypiticus Firefly

5 Vibrio fischeri common bioluminescent bacteria in photophores (light organs) of marine organisms common bioluminescent bacteria in photophores (light organs) of marine organisms Gram negative, f. Vibrionaceae Gram negative, f. Vibrionaceae pathogenic and symbiotic interactions with animal tissuepathogenic and symbiotic interactions with animal tissue virulent pathogens of crustaceans, also free living saprophytic cells in seawatervirulent pathogens of crustaceans, also free living saprophytic cells in seawater symbiosis established by inoculation of juvenile animal hostssymbiosis established by inoculation of juvenile animal hosts

6 V. fischeri streak plate

7 the Lux operon gene group responsible for bioluminescence, synthesizes luciferase, key catalyst gene group responsible for bioluminescence, synthesizes luciferase, key catalyst consists of 8 main genes consists of 8 main genes three parts: regulatory genes, fatty acid reductase polypeptides, and luciferase subunits three parts: regulatory genes, fatty acid reductase polypeptides, and luciferase subunits

8

9 luxR luxI luxCDABEG

10 Luciferase Cycle

11 Protocol in a nutshell extract Vibrio fischeri DNA w/ DNeasy ® Tissue Kit extract Vibrio fischeri DNA w/ DNeasy ® Tissue Kit create genomic library w shotgun cloning create genomic library w shotgun cloning Sal I restriction digest of the chromosome Sal I restriction digest of the chromosome ligate restriction fragments into inducible Promega pGEM® -3Zf(+) vector ligate restriction fragments into inducible Promega pGEM® -3Zf(+) vector transform BL21 (DE3) E. coli w/ cloned vectors transform BL21 (DE3) E. coli w/ cloned vectors select correctly transformed colonies by blue- white screening (and possibly bioluminescence) select correctly transformed colonies by blue- white screening (and possibly bioluminescence) manipulate lux expression in successfully transformed cells manipulate lux expression in successfully transformed cells

12 Why Sal I? cleaves a six base pair palindromal sequence (GTCGAT) w/ sticky ends 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 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 but most importantly… the lux operon exists on a Sal I restriction fragment of around 9kb

13 Why pGEM® -3Zf(+) ? T7Sal I lacZAmp T7Sal I lacZAmp

14 Why BL21 (DE3) E. coli ? laboratory strain with the gene encoding T7 RNA polymerase conveniently under lac operon control laboratory strain with the gene encoding T7 RNA polymerase conveniently under lac operon control induce/repress with carbs or analogs induce/repress with carbs or analogs expression of lux operon through direction of lac operon- E. coli media expression of lux operon through direction of lac operon- E. coli media compatible Shine-Dalgarno sequences compatible Shine-Dalgarno sequences

15 Timeline Week of Sept 13 th – Week of Sept 13 th – 15 pts Week of Sept 20 th – Week of Sept 20 th –15pts Week of Sept 27 th – Week of Sept 27 th –10pts Week of Oct 3 rd – Week of Oct 3 rd –5pts Week of Oct 10 th – Week of Oct 10 th –5pts Until Nov 22 nd - Until Nov 22 nd - receive vector plasmid and DNeasy, begin DNA extraction receive vector plasmid and DNeasy, begin DNA extraction chromosomal and vector digestion, gel verification chromosomal and vector digestion, gel verification ligation and gel verification ligation and gel verification prepare competent cells, transformation, and selection prepare competent cells, transformation, and selection manipulation of operon manipulation of operon possibly redoing steps… possibly redoing steps…

16 Budget Promega pGEM® -3Zf(+) vector $66.00 Promega pGEM® -3Zf(+) vector $66.00 DNeasy Tissue Kit (50) $ DNeasy Tissue Kit (50) $ T4 DNA ligase $33.00 T4 DNA ligase $33.00 Sal I $55.00 Sal I $55.00 Total $264.00

17 References Altman, John. Autoinduction of Expression in the T7 Expression System. Altman Laboratory at Emory Vaccine Center. 3 Sept _annot.html _annot.html _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 fault.html fault.html fault.html Promega Bacterial Expression Vectors. Promega Corporation. 3 Sept Slock, James. Molecular Biology Experiments Utilizing the lux Genes of Vibrio fischeri and gfp Gene of Aequoria victoria. King’s College PA. 3 Sept Slock, James. Molecular Biology Experiments Utilizing the lux Genes of Vibrio fischeri and gfp Gene of Aequoria victoria. King’s College PA. 3 Sept Winfrey, Michael, Marc Rott, and Alan Wortman. Unraveling DNA Molecular Biology for the Laboratory. New Jersey: Prentice Hall, 1997.


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