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 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: