Genetic Engineering BSC 1010L Transformation of E. coli with Jellyfish GFP.
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Transcript of Genetic Engineering BSC 1010L Transformation of E. coli with Jellyfish GFP.
What is transformation?
Transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation, and expression of exogenous DNA taken up from the cell’s surroundings
Plasmid
Bacterial chromosomal
DNA
Cell wall
Escherichia coli
It has become an important research organism for molecular biology
Growth requirements are well characterized
E coli is the most common bacterium in the human gut
A single microscopic cell can divide to form a visible colony with millions of cells overnight
Molecular biology of E coli is well understood
Has been extensively studied
Genome has been sequenced
Many strains commercially available
Reproduces very rapidly
Bioluminescence – the production and emission of light by a living organism
This phenomenon found in fungi and many animals including fireflies and other insects, marine invertebrates and vertebrates
Blackdragon fish
Mushrooms
Firefly
Dinoflagellates
In the jellyfish, Aequorea victoria, a greenish biolumunescence results from the activity of Green Fluorescent Protein (GFP)
GFP is composed of 238 amino acid residues that exhibits a bright green fluorescence (509 nm) when exposed to blue light (395nm)
The Transformation Plasmid
The GFP gene has already been inserted into the pGLO plasmid.
A restriction enzyme was used to cut out the GFP gene in jellyfish DNA
The same restriction enzyme was used to cut open the pGLO plasmid
The GFP gene fragment and pGLO plasmid were incubated with DNA ligase
The recombinant pGLO plasmid is now ready for use
The pGlo plasmid
– Beta Lactamase• Provides ampicillin
resistance
– araC regulator protein• Regulates GFP
transcription
– Green Fluorescent Protein• Aequorea victoria jellyfish
gene
pGLOori
blaGFP
araC
How does it work?
GFP
Beta lactamase(ampicillin resistance)
pGlo Plasmids
Bacterial chromosomal DNA
Transform bacteria with the pGlo plasmid and grow under various conditions
Ampicillin Action and Resistance
Antibiotics have various methods of interfering with bacterial growth: inhibiting cell wall biosynthesis or blocking protein synthesis
Ampicillin inhibits peptidoglycan synthesis: the cell wall polymer consisting of sugars and amino acids
The ampicillin resistance protein, β-lactamase, cleaves the β-lactam ring of ampicillin molecules, which leaves them unable to interfere with peptidoglycan synthesis
Transformation Procedure: Overview
• Suspend bacterial colonies in Transformation Solution
• Add pGLO plasmid DNA
• Place tubes on ice
• Heat shock at 42oC and place back on ice
• Incubate with LB nutrient broth
• Streak plates
Why perform each step?
CaCl2 treatment on ice crystallizes fluid membranes and stabilizes distribution of charged molecules
CaCl2 Transformation solution provides Ca++ cations that neutralize the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane, allowing the DNA to enter the cells
Ca++
Ca++
OCH2
O
P O
O
O Base
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
OCa++
Why perform each step?
Heat-shock increases permeability of cell membrane
Luria-Bertani Nutrient broth incubation allows beta lactamase expression
Beta lactamase(ampicillin resistance)
pGlo Plasmids
Bacterial chromosome DNA
Cell wall
Selection for Transformants
• Grow transformed bacteria under various conditions
• On which plates will colonies grow?
• On which plates will colonies glow?
The pGlo SystemAreas of Special
Attention
Timing is important…be
efficient!!
Mix contents before pipetting!!!
A film of plasmid must be on the
loop!
1 – LB/AMP/Ara plate
Supplies (each lab group):
2 – micro test tubes
Micro tube rack
Sterile transfer pipette
Sterile inoculation loop
2 – LB/AMP plates
1 – LB plate
Step 4: Heat Shock Bacteria:
It is very important to directly transfer tubes from the ice to the water bath and then directly back to the ice after 50 seconds have elapsed.