Transcript GENETIC MODIFICATION and pGLO

GENETIC
ENGINEERING
BIOLOGY 2/2A
MOTZKO
Flavr Savr
(1994)
Combined tomato
antisense (mutated)
DNA to prevent the
ripening process
GENETIC ENGINEERING
Recombinant
DNA = utilizing DNA from two
different sources
Transgenic DNA = utilizing DNA from two
different species
Hence,
all transgenic DNA is recombinant
but not all recombinant DNA is transgenic
Fast Growing
Salmon (2010)
Combines eel
pout and salmon
DNA to produce
fish that grows 2x
fast
A Cleaner
Pig Sty
(2012)
Combines
E.coli, mouse
and pig DNA to
produce swine
that produce
70% less
phosphorus
waste
And A Less
Gassy Cow
(2009)
Bioengineered
cows produce
25% less
methane gas as
a byproduct of
rumination
Venomous
Cabbage
(2002)
Combines
scorpion and
cabbage DNA to
create vegetable
that is poisonous
to caterpillars but
not to humans
A Banana A
Day…
Banana saplings
infected with virus
carrying DNA codes
for immunity to
Hepatitis B.
Vaccine then
passed to humans
via eating bananas
The Incredible,
Edible Egg (2007)
Bioengineered
British chickens lay
eggs that contain
monoclonal
antibodies used to
fight arthritis and
multiple sclerosis
Evergreen (or
not so green)
(2003)
Colorado State
bioengineers
evergreens that
change color when
under attack from
insects or when
environmentally
stressed.
Biosteel
(2000)
University of Wyoming
Study combines DNA
of orb-weaving
spiders with goats to
produce large
quantities of spider-silk
in lactating females.
Fluorescent
Feline Clones
(2007)
South Korean experiment
seeks to utilize
bioluminescence to show
that gene expression can
be regulated.
If so, felines could be
created that express
human diseases for
research purposes.
WHAT?
A
bacterial transformation
Forcing a bacterial cell to
transcribe and translate a
sequence of foreign DNA
into protein
WHY?
To
create a transgenic organism (contains
DNA from two different species)
To show how genes are organized,
controlled and expressed
To demonstrate how artificial selection
affects the evolution of a population
WHO?
Bacteria:
Source
Escherichia coli HB101
of Foreign DNA:
Aqueoria victoria
(naturally bioluminescent jellyfish)
Aequorea victoria
Why Escherichia coli?
Prokaryotic
No
introns
Rapid growth
Non-pathogenic
HOW?
The
jellyfish gene for
bioluminescent protein (GFP) has
already been spliced into a ring of
DNA called a plasmid
E.coli
forced to absorb these
plasmids through a combination of
chemicals and heat shock
Contents of pGLO Plasmid

Amp = gene coding for
beta lactamase
(makes E.coli HB101
resistant to the
antibiotic ampicillin)

araC = gene coding for
arabinase, an enzyme
that allows E.coli HB101
to metabolize the sugar
arabinose

GFP = gene that codes
for Green Fluorescent
Protein
pGLO Operons

Beta Lactamase = codes for
an enzyme that breaks
down the antibiotic
ampicillin

Arabinase = codes for
enzyme which helps
hydrolyze the sugar
substrate arabinose
How Do We Get The pGLO Plasmid
Into E.coli HB101?
Four Testing Groups
(Control & Independent Variables)
 Four
Test Groups Plates
1
LB (luria broth)
2
LB + Ampicillin (antibiotic)
1
LB + Ampicillin + Arabinose (sugar)
Contents of Each Vial

Vial 1 (for LB – and
LB/amp-) gets 1
loop of bacteria,
transformation
solution but NO
pGLO

Vial 2 (for LB/amp +
and LB/amp/ara +)
gets 1 loop of
bacteria,
transformation
solution and pGLO
Transformation Solution
Bacterial
plasma
membrane will
have its semipermeability
altered through use
of dilute CaCl2
solution
Heat Shock
42
degrees C
for 50 seconds,
then back to
ice
Luria Broth & Plating
Supply cells with Luria broth
for recovery from stress
 After recovery, pour half of
DNA – vial onto LB- and half
onto LB/amp –
 Pour half of DNA + vial onto
LB/amp+ and half onto
LB/amp/ara+ plate

Incubation
Incubate
at
enteric (37 C)
temperatures
for 24-72 hours
HOW IS OUR
GENOME A
BUREAUCRACY?
Operon System

A series of structural and
regulatory genes arranged in a
manner such as to produce
various proteins only when
needed by the cell

A more efficient way to arrange
and manage gene expression

Only 3-5% of genes need to be
expressed at any given time
Operon Components
 Promoter
Region = Place where RNA
polymerase binds to DNA strand to start the
transcription of genes into mRNA
 Operator: Location adjacent to promoter where
repressor protein binds, blocking RNA
polymerase from binding to the promoter
 Structural Genes: The genes that are transcribed
when RNA polymerase can bind to the
promoter region
 Repressor Gene: codes for repressor protein to
block the operator region
Inducible System
 Inducible
System:
structural genes can
only be transcribed
when a specific
substrate is present
 Ex:
Lactose operon
(Jacob & Monod)
What Is Required To Induce Each
Operon To Code For Its Protein?
Operon Function
When the substrate
is absent, the
repressor protein is
produced and
blocks access to
the promoter of the
operon
 When the substrate
is present, the
repressor is inhibited
and the unblocked
promoter can be
accessed

If Function Dictates Form, How Can
Recombinant DNA Be Expressed?
Antibiotic Selection Process
 Not
all bacteria will absorb the plasmids. We will kill
off those that fail to absorb the plasmids by placing
some of bacteria in environments containing the
antibiotic ampicillin.
 Those bacteria that survive must have picked up the
pGLO plasmid because it contains the gene betalactamase, which destroys the antibiotic.
Inducing GFP Production
Not all the bacteria that have absorbed the
pGLO plasmid will produce GFP.
 To induce/force the bacteria to produce GFP,
they must also be forced to produce the
enzyme arabinase.
 Why? Because the GFP gene was inserted into
the plasmid adjacent to the arabinase gene.
 If you “turn on” the arabinase gene, you will
“turn on” the GFP gene by default.

Transformation Efficiency
 Def:
How many E. coli cells picked up the
pGLO plasmid relative to the amount of
pGLO plasmid to which they were
exposed (expressed in trasnformants/µg)
 Calculate transformation efficiency for LA+
and LAA+ plates (as they were the only
ones exposed to plasmid)
 Use pp. 43-48 in your pGLO manual