Bacterial Transformation Lab “pGLO”

pGLO• A fluorescent protein

from the jellyfish, Aequorea victoria

• The pGLO plasmid contains several genes that are necessary for producing and expressing the pGLO protein in whatever organism it is found in.

Other Glowing Critters

What is Bacterial Transformation?

• Bacterial cells taking up DNA from their environment.

• Discovered by Frederick Griffith.

Bacterial Transformation Lab

1. Bacterial Cells and plasmid DNA are mixed.

2. Cells take up plasmid.

3. Cell/DNA mix is plated on nutrient agar with antibiotic.

4. Only cells which obtained plasmid DNA will grow…and glow!

What is a plasmid?

ori

bla

• Small circular DNA molecule.

• Replicates autonomously • Originally evolved in

bacteria• May contain antibiotic

resistance gene or be modified to contain other genes.– bla is an ampicillin

resistance gene

Bacterial Cells and DNA

Chromosomal DNA

Chromosomal

Bacterial cell

Plasmid DNA

pGLO

blaGFP

pGLO Plasmid: Most Important

Components• bla gene– Bacteria with this gene

produce beta lactamase, an enzyme that allows them to grow in the presence of ampicillin

• GFP gene– Bacteria with this gene

glow under UV light

Reasons for Each Transformation Step

CaCl2 treatment Positive charge of

Ca2+ ions neutralizes: • negative charge of

DNA phosphates • negative charge of

membrane phospholipids

Ca++

Ca++

OCH2

O

P O

OO Base

CH2

O

P

OO

O

Base

OH

Sugar

Sugar

OCa++

Incubation on iceslows fluid cell membranes

Heat-shockincreases permeability of cell membrane

Nutrient broth incubationallows beta lactamase expression prior to

exposure Growth on agar containing

arabinoseinitiates transcription of GFP genes

(modified operon)

Reasons for Each Transformation Step

Transformation Results

Only cells getting pGLO plasmid grow and glow

Only cells getting pGLO plasmid grow

Without pGLO plasmid, nothing can grow

All cells grow since there is no antibiotic on the plate