Transformation of Escherichia coli Using an Inducible .ppt

1、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 speciesfungi,

2、 insects, algae, free living bacteria, mollusks, crustaceans, and other animals in symbiosis with bioluminescent bacteria,Evolutionarily speaking,based upon reproductive communication and competitionattract mates or advertise high fitness levels (remember energy allocation from EvoEco?)illumination

3、for predation or protection ex. fireflies, cuttlefish, dragonfishor just to look cool,Dragonfish,Comb Jelly,Panellus stypiticus,Firefly,Vibrio fischeri,common bioluminescent bacteria in photophores (light organs) of marine organisms Gram negative, f. Vibrionaceae pathogenic and symbiotic interaction

4、s 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

5、main genesthree 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

6、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

7、palindromal sequence (GTCGAT) w/ sticky endsrestriction fragment length of 4000 bp from average genome, but this may vary due to G+C contentbut 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 ?,laborator

8、y 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 ptsWeek of Sept 20th 15ptsWeek of Sep

9、t 27th 10ptsWeek of Oct 3rd 5ptsWeek of Oct 10th 5ptsUntil 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 redoin

10、g steps,Budget,Promega pGEM -3Zf(+) vector $66.00 DNeasy Tissue Kit (50) $110.00 T4 DNA ligase $33.00 Sal I $55.00Total $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/

11、altman/f_protocols/f_tetramers/autoind_annot.htmlBluth, 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.htmlPromega Bacterial E

12、xpression Vectors. Promega Corporation. 3 Sept. 2004. http:/ James. Molecular Biology Experiments Utilizing the lux Genes of Vibrio fischeri and gfp Gene of Aequoria victoria. Kings College PA. 3 Sept. 2004. Winfrey, Michael, Marc Rott, and Alan Wortman. Unraveling DNA Molecular Biology for the Laboratory. New Jersey: Prentice Hall, 1997.,