Transcript O - morescience

Protein Transformation Lab Intro
UCSD: BioBridge Program
E. coli
By Lynn More - Olympian High
School along with UCSD
What is Transformation?
Bacterial
chromosome
Uptake of foreign DNA,
often a circular plasmid
Plasmid
Bacterial
chromosome
Plasmid
Bacteria now express cloned fluorescent protein
(transcription of gene and translation of mRNA to
protein at ribosomes).
Allow bacteria to grow for 1-3
days on plate with ampicillin.
What is protein transformation?
Introducing DNA that expresses preferred gene(s) into a host.
Why do protein transformation
1. Inhibit or silence the expression of a gene
i.e. - allows you to determine its function or importance
2. Carry out certain functions
i.e. - make insulin, clot blood, resist pests, resist antibiotics, eat oil
3. Used as markers to track the location and function of the gene
i.e. - fluorescent proteins
Aequorea victoria and Discovery of GFP1960’s
OSAMU SHIMOMURA
Co-winner of Nobel Prize
There are three amino acids
which are critical for GFP’s
green fluorescent color.
Only a 1 amino acid difference
changes green to blue, and
blue to cyan.
Fluorescent Proteins-Applications
•Transgenic Mice
Neuron
Transgenic Zebra Fish
Roger Tsien and Rainbow Proteins
17 Mut
mgrape
1
6 Mut
DsRed.T
1
33 Mut
Dimer 2
6 Mut
3 Mut
mHoneydew
mCherry
mRFP1
4 Mut
8 Mut
3 Mut
7 Mut
mStrawberry
mBanana
mOrange
mTangerine
Cellular organelles “marked” with FPs
Human cell stained with two different
fluorescent proteins to visulalize
cytoskeletal components. Transfected
with GFP-tubulin / mCherry actin
(Ben Giepmans)
C Elegans transfected with GFP
tubulin construct (Susan Kline)
The rainbow of mFruit Fluorescent Proteins
http://www.dnai.org/a/index.html
Central Dogma
DNA--->
mRNA--->
Protein--->
Trait
Materials

4 microcentrifuge tubes

2 Empty (clear/colorless)

CaCl2 on ice (blue)

either PM1 or PM2 on ice. (clear/colorless)

4 disposable transfer pipette

1 Inoculating loops

3 Q-Tips

LB plates (2 LB/Amp - red line, 1 LB no Amp)

Lab station waste containers - one with 10% bleach, other empty
Equipment:

42˚C for 45 seconds
Vortex
 Hot water bath
 Incubator
37˚C / 98.6˚F
Human body temperature
Label your equipment
LB/AMP +
P5
Table ?
A+
LB/AMP -
P5
LB/No AMP -
P5
Table ?
A-
Table ?
What is a plasmid?
•
•
PM means Plasmid Mix
What: A small circular piece of
DNA naturally occurring in
bacteria
PM1
Green
Blue
Grape
Why: Can be altered in lab to
express protein of interest.
PM2
Cherry
Tangerine
Banana
promoter
GFP
Origin
Gene of Interest:
AMPR - Ampicillin Resistance Gene
Antibiotic Resistance
AmpR
Stop
Gene of Interest:
Fluorescent Protein
Escherichia coli
• What?
• AKA (also known as) E. coli
• Prokaryote
• Single-celled organism
• No nucleus
• No membrane –bound organelles
• Why?
• Small, so only need…
• Food (LB Agar)
• Little space
• Warm temps (37˚C)
• Little humidity
•
•
•
Reproduces fast
• Binary fission
• x 106
Can uptake foreign DNA
Clones itself & its contents
Why calcium chloride?
• Calcium Chloride (CaCl2)
Transformation solution
• CaCl2 is necessary because:
• The positive charge of Ca++
ions neutralizes DNA’s
slightly negative charge
• increases the diffusion of its
foreign genetic information
through the cell wall and cell
membrane into the bacteria.
Ca++
Ca++
O
O P O
O
CH2
Base
O
Sugar
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
Pipette Techniques
1.0 mL
• Squeeze the bulb with two fingers - firmly - hold
• Insert into the fluid
• Gently release SOME pressure on the bulb!
Until the liquid gets to the 0.1 m mark
0.5 mL
• Maintain the pressure
If you release all pressure the
0.25 mL
fluid will be sucked up into the
bulb rather than to the
measurement line.
0.1 mL
• Move the pipette to the microcentrifuge tube
• Squeeze the fluid from the pipette into the tube.
• Repeat, measuring to 0.25 mL
• Add the liquid to the microcentrifuge tube
• Repeat measuring 0.5 mL, then
• Add the liquid to the microcentrifuge tube
• Move 1.0 mL into the pipette then to the microcentrifuge tube
• Cap, Mix with rocking & vortex, empty and returnL
The what’s and why’s of Heat Shock
•
Incubate on ice (10 minutes) slows
fluid cell membrane (constricts pore
size)
•
Heat-shock (42˚C for 45 seconds)
increases permeability of
membranes (dilates/opens pores,
allowing the plasmid to get inside the bacteria)
•
Incubate on ice (2 minutes) slows
fluid cell membrane (reduces
permeability again, “locking” the plasmid
inside the bacteria)
Plate Streaking Techniques
Purpose is to spread out the bacteria so it has access to more food and space
Plasmid = a vector that carries genetically engineered DNA segment into a host cell.
Recombinant DNA
Insert the DNA (plasmid)
Using a Heat Shock Method
Bacteria
cell
Bacterial
chromosome
Bacteria plated on
LB agar + antibiotic
cloning
X 106
Collect culture
Only bacteria containing
Recombinant DNA grow
DNA
Purification
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
Satellite Colonies
Regular bacteria wh
Are eating the food t
has been treated wit
the enzyme - and no
Not poisonous to the
Genetically Modified
E. Coli Bacteria
Region around
Modified Bacteria
where enzyme has
Broken down the antibiotic
Agar
What’s happening in the petri dish?
Represents _________________________________________
LB Agar - a nutrient substrate to encourage growth
Represent ___________________________________________
Ampicillin - an antibiotic that inhibits bacterial growth
Represent ______________
Bacteria growth
Bacteria killed by ampicillin
______________________
Represent _________________________________
Genetically transformed bacteria that are:
1. Resistant (or shielded) from the effects of ampicillin
2. Marked with a Fluorescent Protein
Ampicillin acts as a __________________
selection mechanism that only allows
___________
transformed bacteria to _____
grow on the plate
Transformation Prep.ppt
Make two Plasmids
Glue/Tape
Glue/Tape
Pick a Fluorescent Protein gene to insert

Restriction Enzyme
mCherry
mTangerine
mBanana
GFP
BFP
Green
Fluorescent
Protein
Blue
Fluorescent
Protein
mGrape
mPlum
Use DNA Ligase (tape or glue) to bond the gene of interest
Restriction Enzyme

Restriction Enzyme
Add an antibiotic resistance gene to both plasmids you make.

Restriction Enzyme
AmpR
gene
TetraR
gene
KanR
gene
PenR
Ampicillin
Tetracycline
resistance
Kanamycin
resistance
Penicillin
resistance
resistance
gene
Time to make
the second
Plasmid Model

Restriction Enzyme

Use DNA Ligase (tape or glue) to bond the gene of interest

Restriction Enzyme
Tuck
Under &
Glue/Tape
Pick a gene of interest to add to the second plasmid you make
Insulin
gene
Save a
diabetic
Factor
VIII
gene
Save a
hemophiliac
Pest
Resistance
Oil Spill
gene
gene
Save a plant
Save an
environmen
t
Congratulations
scientists you
have just made
recombinant
DNA: genetically
engineered DNA
with genes that
can save lives!
Tuck
Under
&
Glue/Tape
Make a Plasmid Activity #?
1. Cut the DNA with a _______________ (Scissors)
2. My gene of interest was (FP - ________ & __________)
3. My goal is to (FP) - track ____________; save ________
4. The petri dish would have:
___________ antibiotic; ___________ antibiotic
so…I need to make the transformed bacteria
resistant to that antibiotic (_____); (_____)
5. What I have made are 2 small circular pieces of DNA
with two genes of interest each & they called plasmids.
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
The plasmids we have…
The plasmid serves as a carrier or transporter of a
genetically engineered DNA segment into a host cell.
Area of Interest - Fluorescent Protein
Gene for
antibiotic
resistance
Restriction Enzyme
Restriction Enzyme
Cuts the DNA
Foreign DNA
Sticky ends help attach to the plasmid
DNA Ligase
Recombinant DNA
EcoRI (pronounced "eco R one") is a commonly used restriction enzyme
isolated from certain strains of E. coli used to cut DNA at specific locations.