Download Transformation of Escherichia coli Using an Inducible

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.