Download pGLO Lab Protocol

pGLO Bacterial Transformation
Green Fluorescent Protein or GFP
What is transformation?
Uptake of foreign DNA, often a circular DNA
called a plasmid
GFP
Bacterial
chromosomal DNA
Amp
Resistance
pGLO plasmids
What is a plasmid?
• A plasmid is a small circular piece of DNA (about 2,000 to
10,000 base pairs) that contains important genetic
information for the growth of bacteria.
A plasmid containing resistance to an
antibiotic is used as a vector.
• The gene of interest is inserted into the vector
plasmid and this newly constructed plasmid is then
put into E. coli that are sensitive to ampicillin.
• Vector - Something that is used to transfer
something else (a mosquito is a vector for the
organism that causes malaria)
The bacteria are then spread over an
agar plate that contains ampicillin.
• The ampicillin provides a selective pressure
because only bacteria that have acquired the plasmid
can grow on the plate.
• Therefore, as long as you grow the bacteria in
ampicillin, it will need the plasmid to survive and it
will continually replicate it, along with your gene of
interest that has been inserted to the plasmid.
Mouse under blue light (left)
Same mouse under normal light (right)
Mouse blood vessels (green-GFP) in tumor (red-DsRed). Mouse with brain tumor
expressing DsRed.
Three 60 day old kittens. Two have been genetically
modified to make red fluorescent protein. All three look
similar under normal light, but when irradiated with blue
light only the two genetically modified kittens glow red.
What is the GFP gene?
• GFP is a green fluorescent protein that normally is found in
jellyfish.
Engineering Green Fluorescent Protein (GFP)
• GFP has been added to :
– rabbits
– rats
– mice
– frogs
– flies
– worms
– and countless other living things
S. Stevens
6/14/2006
GFP requires the sugar Arabinose in
order to turn on the GFP Gene.
• Called the arabinose operon of E.coli:
• Arabinose, a 5 carbon sugar, serves as
energy and carbon source for the
bacteria.
• There are 3 genes (araB, araA, &
araD) that need to be expressed for
the transport and breakdown of
arabinose.
• These genes are only expressed when the
sugar arabinose is present in the environment.
These three ara genes are clustered together
and make up the arabinose operon.
The ara regulatory sequences precede the 5’ end
of the araB gene.
• They are “upstream” from the coding sequences.
• The promoter region is the site that binds the RNA
polymerase enzyme (required for transcription).
• Just upstream from the promoter lies the binding site for the
arabinose gene activator, araC.
• This activator protein is bound to the regulatory sequence
and changes shape when the sugar, arabinose, binds to it.
• This shape change allows RNA polymerase to bind
at the promoter sequence & transcription is initiated.
• The three genes, araB, araA, and araD are
transcribed and translated to produce the three
enzymes required to breakdown arabinose.
• As the arabinose is used up and broken down,
AraC has no arabinose to bind to it, AraC returns
to its original shape, RNA polymerase binding
does not occur and transcription is then turned off.
Overview:
• A. AraC is blocking the
binding of RNA polymerase
for transcription of the gene
responsible for degradation
of arabinose.
• B. Arabinose binds to araC
causing a change that
reveals the promoter region.
• C. RNA polymerase binds
to promoter.
• D. Transcription of the
arabinose operon is
achieved.
pGLO Plasmid
Cloning of Plasmid
TRANSFORMATION
• THE PROCESS BY WHICH BACTERIA CELLS
PICK UP AND INCORPORATE DNA FROM
DEAD BACTERIA CELLS.
• THEY TAKE UP DNA FROM DEAD CELLS OF
THE SAME OR A CLOSELY RELATED SPECIES
Genomic Bacterial DNA
S. Stevens
6/14/2006
Plasmid
S. Stevens
6/14/2006
Antibiotic Resistance
Amp resistant E. Coli on Left
E. Coli on Right
S. Stevens
6/14/2006
Competent Cells
• Cells must be made competent
before they can absorb the plasmids
that carry the new genes.
• In this lab, we will make our cells
competent by soaking them in CaCl2
solution and heat shocking them.