Transformation of Transformation of Escherichia coliEscherichia coli Using an Using an

Inducible Expression Vector Inducible Expression Vector Containing the BioluminescentContaining the Bioluminescent

Vibrio fischeriVibrio fischeri LuxLux Operon Operon

byby

Bryan Hart & Crystal HarmonBryan Hart & Crystal Harmon

BioluminescenceBioluminescence

biologically mediated synthesis of biologically mediated synthesis of compounds that react to emit visible compounds that react to emit visible light energylight energy

found in diverse range of speciesfound in diverse range of species

fungi, insects, algae, free living fungi, insects, algae, free living bacteria, mollusks, crustaceans, and bacteria, mollusks, crustaceans, and other animals in symbiosis with other animals in symbiosis with bioluminescent bacteriabioluminescent bacteria

Evolutionarily Evolutionarily speakingspeaking

based upon reproductive communication based upon reproductive communication and competitionand competition

attract mates or advertise high fitness attract mates or advertise high fitness

levels (remember energy allocation from levels (remember energy allocation from EvoEco?)EvoEco?)

illumination for predation or protection illumination for predation or protection

ex. fireflies, cuttlefish, dragonfishex. fireflies, cuttlefish, dragonfish

or just to look coolor just to look cool

Dragonfish Comb Jelly

Panellus stypiticus Firefly

Vibrio fischeriVibrio fischeri

common bioluminescent bacteria common bioluminescent bacteria in photophores (light organs) of in photophores (light organs) of marine organismsmarine organisms

Gram negative, f. Vibrionaceae Gram negative, f. Vibrionaceae • pathogenic and symbiotic pathogenic and symbiotic

interactions with animal tissueinteractions with animal tissue• virulent pathogens of crustaceans, virulent pathogens of crustaceans,

also free living saprophytic cells in also free living saprophytic cells in seawaterseawater

• symbiosis established by inoculation symbiosis established by inoculation of juvenile animal hostsof juvenile animal hosts

V. fischeriV. fischeri streak plate streak plate

the the LuxLux operon operon

gene group responsible for gene group responsible for bioluminescence, synthesizes bioluminescence, synthesizes luciferase, key catalyst luciferase, key catalyst

consists of 8 main genesconsists of 8 main genes

three parts: regulatory genes, three parts: regulatory genes, fatty acid reductase polypeptides, fatty acid reductase polypeptides, and luciferase subunitsand luciferase subunits

luxluxR R luxluxII

luxluxCDABEGCDABEG

Luciferase CycleLuciferase Cycle

Protocol Protocol in a nutshellin a nutshell

extract extract Vibrio fischeriVibrio fischeri DNA w/ DNeasy DNA w/ DNeasy®® Tissue Tissue KitKit

create genomic library w shotgun cloningcreate genomic library w shotgun cloning• Sal Sal I restriction digest of the I restriction digest of the

chromosome chromosome • ligate restriction fragments into ligate restriction fragments into

inducible Promega pGEM® -3Zf(+) inducible Promega pGEM® -3Zf(+) vectorvector

• transform BL21 (DE3) transform BL21 (DE3) E. coli E. coli w/ cloned vectorsw/ cloned vectors

• select correctly transformed colonies by select correctly transformed colonies by blue-white screening (and possibly blue-white screening (and possibly bioluminescence)bioluminescence)

manipulate manipulate lux lux expression in successfully expression in successfully transformed cellstransformed cells

Why Why Sal Sal I?I? cleaves a six base pair palindromal cleaves a six base pair palindromal

sequence (GTCGAT) w/ sticky endssequence (GTCGAT) w/ sticky ends

restriction fragment length of 4000 bp restriction fragment length of 4000 bp from average genome, but this may from average genome, but this may vary due to G+C contentvary due to G+C content

but most importantly… the lux operon but most importantly… the lux operon exists on a exists on a Sal Sal I restriction fragment of I restriction fragment of around 9kb around 9kb

Why pGEM® -3Zf(+)Why pGEM® -3Zf(+) ??

T7T7 Sal Sal II laclacZZ AmpAmp

Why BL21 (DE3) Why BL21 (DE3) E. E. coli coli ??

laboratory strain with the gene laboratory strain with the gene encoding T7 RNA polymerase encoding T7 RNA polymerase conveniently under conveniently under laclac operon control operon control

induce/repress with carbs or analogsinduce/repress with carbs or analogs expression of expression of luxlux operon through operon through

direction of direction of laclac operon- operon- E. coliE. coli media media compatible Shine-Dalgarno sequencescompatible Shine-Dalgarno sequences

TimelineTimeline Week of Sept 13Week of Sept 13thth – –

15 pts15 pts

Week of Sept 20Week of Sept 20thth – –15pts15pts

Week of Sept 27Week of Sept 27thth – –10pts10pts

Week of Oct 3Week of Oct 3rdrd – –5pts5pts

Week of Oct 10Week of Oct 10thth – –5pts5pts

Until Nov 22Until Nov 22ndnd--

receive vector receive vector plasmid and DNeasy plasmid and DNeasy , begin DNA , begin DNA extractionextraction

chromosomal and chromosomal and vector digestion, gel vector digestion, gel verificationverification

ligation and gel ligation and gel verificationverification

prepare competent prepare competent cells, cells, transformation, and transformation, and selectionselection

manipulation of manipulation of operonoperon

possibly redoingpossibly redoing steps…steps…

BudgetBudget

Promega pGEM® -3Zf(+) vector Promega pGEM® -3Zf(+) vector $66.00$66.00

DNeasy Tissue Kit (50)DNeasy Tissue Kit (50) $110.00$110.00

T4 DNA ligaseT4 DNA ligase $33.00$33.00 SalSal I I $55.00$55.00

Total $264.00Total $264.00

ReferencesReferencesAltman, John. Altman, John. Autoinduction of Expression in the T7 Expression Autoinduction of Expression in the T7 Expression

SystemSystem. Altman Laboratory at Emory Vaccine Center. 3 Sept. . Altman Laboratory at Emory Vaccine Center. 3 Sept. 2004. 2004. http://www.microbiology.emory.edu/altman/f_protocols/f_tetramehttp://www.microbiology.emory.edu/altman/f_protocols/f_tetramers/autoind_annot.htmlrs/autoind_annot.html

Bluth, Brian J., Sarah E. Frew, and Brian McNally. Bluth, Brian J., Sarah E. Frew, and Brian McNally. Cell-Cell Cell-Cell Communication and the Communication and the lux lux operon in operon in Vibrio fischeriVibrio fischeri. Carnegie . Carnegie Mellon University. 3 Sept. 2004. Mellon University. 3 Sept. 2004. http://www.bio.cmu.edu/courses/03441/TermPapers/97TermPapehttp://www.bio.cmu.edu/courses/03441/TermPapers/97TermPapers/lux/default.htmlrs/lux/default.html

Promega Bacterial Expression VectorsPromega Bacterial Expression Vectors. Promega Corporation. 3 . Promega Corporation. 3 Sept. 2004. Sept. 2004. http://http://www.promega.com/vectors/bacterial_express_vectors.htmlwww.promega.com/vectors/bacterial_express_vectors.html

Slock, James. Slock, James. Molecular Biology Experiments Utilizing the lux Genes Molecular Biology Experiments Utilizing the lux Genes

of of Vibrio fischeriVibrio fischeri and gfp Gene of  and gfp Gene of Aequoria victoriaAequoria victoria. King’s . King’s College PA. 3 Sept. 2004. College PA. 3 Sept. 2004. <http://www.kings.edu/biology/lux/luxbiolum.html> <http://www.kings.edu/biology/lux/luxbiolum.html>

Winfrey, Michael, Marc Rott, and Alan Wortman. Winfrey, Michael, Marc Rott, and Alan Wortman. Unraveling DNA Unraveling DNA Molecular Biology for the LaboratoryMolecular Biology for the Laboratory. New Jersey: Prentice Hall, . New Jersey: Prentice Hall, 1997.1997.