bacterial transformation training. a n e legant w ay to s tudy d evelopmental n eurobiology ( with a...
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BACTERIAL TRANSFORMATION BACTERIAL TRANSFORMATION TRAININGTRAINING
AN ELEGANT WAY TO STUDY DEVELOPMENTAL
NEUROBIOLOGY (WITH A LITTLE HELP FROM GFP)
Gloriana Gallegos TrujilloJin Lab
WE CAN VISUALIZE THE C. ELEGANS NERVOUS SYSTEM USING FLUORESCENT PROTEINS
nose
tail Bessereau Lab
“brain”
WHY DO WE USEGREEN 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
6
Drosophila– most common model organism
E. coli – easy to transform
• Easy to manipulate for experiments • Short life-cycle• Easy to keep alive• Short generation times
C. elegans - genetically mapped and able to target specific cells for study
Lab mouse – easy to alter genetically
OUR MODEL ORGANISM: C. ELEGANS
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
reproductive, digestive, nervous, excretory
eggs
sperm
ADULT HERMAPHRODITE LIES ON ITS SIDE AND MOVES IN A WAVE PATTERN
A SIMPLIFIED SYNAPSE: HOW NEURONS COMMUNICATE
target cell
presynaptic neuron
fusion zone
vesicles loaded with neurotransmitter
chemical signal
electrical signal
my thesis topic
synaptic cleft
GFP AS A TOOL FOR VISUALIZING WHICH CELLS PROTEINS ARE
PRESENT IN
Puev-3-GFP
Trujillo, et al. unpublished data.
p
HOW DO WE GET DNA INTO THE WORM?
BY INJECTING WORM GONADS
A PROTEIN IN SYNAPTIC VESICLES IS LABELED WITH GFP
muscle cells
synaptobrevin-GFP
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 unpublished data.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 ACTIVITIESFLUORESCENT PROTEIN ACTIVITIES
Bacterial Transformation
Protein Purification
Discovery of GFP-Discovery of GFP-1960’s1960’sAequorea victoriaAequorea victoria
OSAMU SHIMOMURA Co-winner of Nobel Prize
www.worldnetcams.com/sealife/cerianthus.jpg
How Fluorescence WorksHow Fluorescence Works
Bioluminescent organism produces its own light.
A fluorescent organism absorbs light at one wavelength (UV) and a re-emits the light at a visible
wavelength= color
Scorpion- UV LightScorpion- Natural Light
http://fireflyforest.net/firefly/2006/11/13/fluorescent-scorpion-in-uv-light/
Natural Light
In the Dark
Bioluminescence Fluorescence
Bioluminescence vs. FluorescenceBioluminescence vs. FluorescenceBioluminescence vs. FluorescenceBioluminescence vs. Fluorescence
www.worldnetcams.com/sealife/cerianthus.jpg
Many organisms have the ability to Many organisms have the ability to fluorescefluoresce
Jellyfish
Amphipod
Spider’s palps
Roger Tsien and Rainbow ProteinsRoger Tsien and Rainbow Proteins
E. COLIE. COLI
What is Transformation?What is Transformation?
Plasmid Uptake of foreign DNA, often a circular plasmid
Bacterial chromosome
What is a 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?What is Transformation?
Plasmid Uptake of foreign DNA, often a circular plasmid
Bacterial chromosome
What is Transformation?What is Transformation?
Plasmid Uptake of foreign DNA, often a circular plasmid
Bacterial chromosome
What is Transformation?What is Transformation?
Bacterial chromosome
Allow bacteria to grow for 1-3 days on plate with ampicillin.
Plasmid Uptake of foreign DNA, often a circular plasmid
Bacterial chromosome
What is Transformation?What is Transformation?
Bacteria now express cloned fluorescent protein…
Bacterial chromosome
Allow bacteria to grow for 1-3 days on plate with ampicillin.
Plasmid Uptake of foreign DNA, often a circular plasmid
Bacterial chromosome
How are plasmids engineered? How are plasmids engineered?
Host DNA fragments (i.e. coral or jellyfish FP coding DNA)
DNA Plasmid Vector
Cut plasmids open with restriction enzymes
Cut genomic DNA into fragments
+
Ligate (paste) fragments into cut DNA vector
Screen for and select plasmid containing FP gene
Bacterial Transformation ProcedureBacterial Transformation Procedure
Shielding the chargeShielding the charge
Ca++
Ca++
OCH2
O
P O
O
O Base
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
OCa++
• CaCl2• Positive charge of Ca++
ions shields negative charge of DNA phosphates
Stress through heatStress 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 is tied to (expressed with) the fluorescent protein gene
• Ampicillin is a selection mechanism that only allows transformed bacteria to grow on the plate
MITOSISa similar process
in diverse species
……...using various organisms to understand humans:
Frog Egg Extract+ sperm DNA
A. DesaiFrog Cell
C.E. Walczak
Marsupial CellS.L. Kline
Fly EmbryoT. Megraw
Frog Egg Extract+ DNA-coated beads
R. Heald
Human CellJ. WatersWorm Embryo
I.M. Cheeseman
Fluorescent Proteins-ApplicationsFluorescent Proteins-Applications
Fluorescent Proteins-ApplicationsFluorescent Proteins-Applications
The rainbow of mFruit Fluorescent ProteinsThe rainbow of mFruit Fluorescent Proteins