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Announcements
New Weekly Schedule
Observer on March 6 and 13
Measuring Zone of Inhibition
Bacterial Transformation
Student Training
By Quanina Quan and UCSD ScienceBridge
Objective
Demonstrate that changes in genotype causes changes
in phenotype by transforming E.coli into fluorescent
E.coli.
Background
What is E.coli?
A rod shaped bacterium found in our lower intestines
What is fluorescence?
A substance that absorbs light at one wavelength (UV) and re-emits light
at a visible wavelength (color)
Background: What is GFP?
Green Fluorescent Protein
Chromophore
How does fluorescence work?
Excited state
Blue light
(High energy)
Green light
(Lower energy)
Ground state
NOTE: FLUORESCENCE
IS NOT LUMINESCENCE
Fluorescence: Absorbs light at
one wave length and emits it at
another
Bioluminescence: Produces its
own light
A Metaphor
Fluorescence: Absorbs light at
one wave length and emits it at
another
Bioluminescence: Produces its
own light
Fluorescent Organisms
Fluorescence: Absorbs light at
one wave length and emits it at
another
Roger Tsien and Rainbow Proteins
GFP
RFP
Uses for FPs
Human Cell
J. Waters
FAQs
Can I make myself or someone I know glow?
Can I get a glowing pet?
Can we turn it on or off?
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?
DNA
RNA
Protein
E. coli bacterial cell
Bacterial genome
Plasmid
Cell produces protein
coded by plasmid DNA.
How We Make E.coli Glow
Plasmid
Uptake of foreign DNA,
often a circular plasmid
Allow bacteria to grow for 1-3
days
DNA  RNA Protein
Bacteria now express cloned fluorescent protein…
What’s on the plasmid
Plasmid Mix 1
Plasmid Mix 2
GFP
Cherry
FP gene
BFP
Tangerine
Grape
YFP
AmpR
Ampicillin
resistance gene
How the Plasmid Gets in the
Bacteria
Show Video
1. Add CaCl2
2. Heat/Shock
Step 0: Label Plates
Step 1: Put CaCl2 in Tube
Step 2: Collect bacterial colonies
“Like scraping whipped cream off of Jello.”
Step 3: Put bacteria in CaCl2
Positive charge of Ca2+ 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
Step 4: Add Plasmid to (+) Tube
Plasmid Mix 1
Plasmid Mix 2
GFP
Cherry
FP gene
BFP
Tangerine
Grape
YFP
AmpR
Ampicillin
resistance gene
Step 5: Heat Shock!
•
Incubate on ice to slow 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.
ICE – HEAT – ICE
Step 6: Plate on Ampicillin
• Ampicillin
inhibits cell wall 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.
We have two controls
LB only (-) = Control 1: check for viable cells
LB amp (-) = Control 2: check for ampicillin function
LB amp (+) = Experimental plate: grow transformants
Why Ampicillin?
LB/AMP +
LB/AMP -
Why Ampicillin?
LB/No Amp
Tricky Parts of Lab
Labeling Plates
Getting Bacteria
Pipetting the right amount
Plating
FAQ
Why don’t I see anything glowing right now?