Download ampicillin resistance

BSC 1010L
Genetic Engineering
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
E coli is the most common bacterium in the
human gut
Has been extensively studied
Growth requirements are well characterized
Genome has been sequenced
Molecular biology of E coli is well understood
It has become an important research organism for molecular biology
Many strains commercially available
Reproduces very rapidly
A single microscopic cell can divide to form a
visible colony with millions of cells overnight
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
Mushrooms
Firefly
Dinoflagellates
Blackdragon fish
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
araC
ori
pGLO
bla
GFP
How does it work?
Transform bacteria with the pGlo plasmid and grow under various
conditions
GFP
Bacterial
chromosomal
DNA
Beta lactamase
(ampicillin
resistance)
pGlo
Plasmids
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 βlactam 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++
O
Ca++
O
P
O
Base
O
CH2
O
Sugar
O
Ca++
O P
O
Base
O
O
CH2
Sugar
OH
Why perform each step?
 Heat-shock increases
permeability of cell
membrane
Cell wall
Bacterial
chromosome
DNA
 Luria-Bertani Nutrient
broth incubation allows
beta lactamase
expression
Beta lactamase
(ampicillin
resistance)
pGlo
Plasmids
Selection for Transformants
• Grow transformed bacteria under various conditions
• On which plates will colonies grow?
• On which plates will colonies glow?
The pGlo System
Areas of Special
Attention
A film of plasmid
must be on the
loop!
Timing is
important…be
efficient!!
Mix contents
before pipetting!!!
Supplies (each lab group):
2 – micro test tubes
Micro tube rack
Sterile transfer pipette
Sterile inoculation loop
1 – LB plate
2 – LB/AMP plates
1 – LB/AMP/Ara plate
Step 1: Add CaCl2 solution to tube:
Step 2: Add competent E. coli to tube:
Step 3: Add pGLO plasmid to +pGLO tube:
Be sure to label the bottom of the petri dish.
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.
Step 5: Plate Bacteria: