Download Uptake of foreign DNA

BACTERIAL TRANSFORMATION
TRAINING
AN ELEGANT WAY TO STUDY
DEVELOPMENTAL
NEUROBIOLOGY
(WITH A LITTLE HELP FROM GFP)
Gloriana Gallegos Trujillo
Jin Lab
WE CAN VISUALIZE THE C. ELEGANS
NERVOUS SYSTEM USING FLUORESCENT
PROTEINS
nose
“brain”
tail
Bessereau Lab
WHY DO WE USE
GREEN FLUORESCENT PROTEIN?



To find out the specific cells
where a protein is made
To find out specific times
during development
proteins are made
To find out what subcellular
location the protein is in: is
it in the nucleus? In the
Golgi?
AN 8-CELL STAGE EMBRYO WITH GFP
LABELING DNA SO WE CAN WATCH
CELLS DIVIDE
Claudiu Giurumescu
MODEL ORGANISMS
Lab mouse – easy
to alter genetically
Drosophila– most
common model
organism
C. elegans - genetically
mapped and able to target
specific cells for study
• Easy to manipulate for experiments
• Short life-cycle
• Easy to keep alive
• Short generation times
E. coli – easy to
transform
6
OUR MODEL ORGANISM: C. ELEGANS
eggs
sperm
reproductive, digestive, nervous, excretory

simple nervous system: hundreds in worms to billions in
humans

old enough to reproduce in 3 days; lifespan is ~2 weeks

small - adults measure 1mm long

transparent or “see through”

hermaphrodites make genetics easy

can be grown in the lab
ADULT HERMAPHRODITE LIES ON ITS SIDE
AND MOVES IN A WAVE PATTERN
A SIMPLIFIED SYNAPSE:
HOW NEURONS COMMUNICATE
electrical
signal
presynaptic neuron
my thesis topic
vesicles loaded with
neurotransmitter
fusion zone
chemical signal
target cell
synaptic cleft
GFP AS A TOOL FOR VISUALIZING WHICH
CELLS PROTEINS ARE PRESENT IN
Puev-3-GFP
p
Trujillo, et al. unpublished data.
HOW DO WE GET DNA INTO THE WORM?
BY INJECTING WORM GONADS
A PROTEIN IN SYNAPTIC VESICLES IS
LABELED WITH GFP
synaptobrevin-GFP
muscle cells
WE CAN USE OUR “MARKER” TO FIND
GENES IMPORTANT FOR SYNAPSE
FORMATION
Nakata, et al. unpublished data.
TEAM JIN/CHISHOLM
Katsu Nakata, RIKEN Japan
FLUORESCENT PROTEIN ACTIVITIES
Bacterial Transformation
Protein Purification
Discovery of GFP-1960’s
Aequorea victoria
OSAMU SHIMOMURA
Co-winner of Nobel Prize
How Fluorescence Works
www.worldnetcams.com/sealife/cerianthus.jpg
Bioluminescence vs. Fluorescence
Bioluminescence
Fluorescence
http://fireflyforest.net/firefly/2006/11/13/fluorescent-scorpion-in-uv-light/
Natural Light
Scorpion- Natural Light
Scorpion- UV Light
In the Dark
Bioluminescent organism
produces its own light.
A fluorescent organism absorbs light
at one wavelength (UV) and a reemits the light at a visible
wavelength= color
Many organisms have the ability to
fluoresce
www.worldnetcams.com/sealife/cerianthus.jpg
Jellyfish
Amphipod
Spider’s palps
Roger Tsien and Rainbow Proteins
E. COLI
What is Transformation?
Bacterial
chromosome
Plasmid
Uptake of foreign DNA,
often a circular plasmid
What is a plasmid?
• A small circular piece of
DNA
• Naturally occurring
• Can be altered in lab to
express protein of interest
What is Transformation?
Bacterial
chromosome
Plasmid
Uptake of foreign DNA,
often a circular plasmid
What is Transformation?
Bacterial
chromosome
Plasmid
Uptake of foreign DNA,
often a circular plasmid
What is Transformation?
Bacterial
chromosome
Uptake of foreign DNA,
often a circular plasmid
Plasmid
Bacterial
chromosome
Allow bacteria to grow for 1-3
days on plate with ampicillin.
What is Transformation?
Bacterial
chromosome
Uptake of foreign DNA,
often a circular plasmid
Plasmid
Bacterial
chromosome
Bacteria now express cloned fluorescent protein…
Allow bacteria to grow for 1-3
days on plate with ampicillin.
How are plasmids engineered?
DNA Plasmid Vector
Host DNA fragments
(i.e. coral or jellyfish FP
coding DNA)
Ligate (paste) fragments into
cut DNA vector
Cut genomic
DNA into
fragments
+
Cut plasmids
open with
restriction
enzymes
Screen for and select plasmid
containing FP gene
Bacterial Transformation Procedure
Shielding the charge
• CaCl2
• Positive charge of Ca++ ions
shields negative charge of
DNA phosphates
Ca++
Ca++
O
O
P
O
O
CH2
Base
O
Sugar
O
Ca++
O
P
O
O
Base
O
CH2
Sugar
OH
Stress through heat
•
Incubate on ice slows fluid
cell membrane
•
Heat-shock increases
permeability of membranes
•
•
Leave in heat 45 seconds!!!
Too short, and bacteria won't
let in plasmid.
Too long, and the bacteria will
die.
•
Why Ampicillin?
• Ampicillin
inhibits cell growth. Only cells that can
inactivate the ampicillin around them will grow.
• Ampicillin resistance
fluorescent protein gene
is tied to (expressed with) the
• Ampicillin is a selection mechanism that only allows transformed
bacteria to grow on the plate
Fluorescent Proteins-Applications
……...using various organisms to understand humans:
MITOSIS
a similar
process
in diverse
species
Fly Embryo
T. Megraw
Marsupial Cell
S.L. Kline
Frog Egg Extract
+ DNA-coated beads
Worm Embryo
R. Heald
I.M. Cheeseman
Human Cell
J. Waters
Frog Cell
Frog Egg Extract
+ sperm DNA
C.E. Walczak
A. Desai
Fluorescent Proteins-Applications
The rainbow of mFruit Fluorescent Proteins