In the pGLO lab, we will:

• Use recombinant DNA

• Genetically engineer E. coli bacteria by inserting a plasmid

• Plate and grow bacteria

• Determine if the proteins are expressed

by genes

Why are bacteria a good choice for genetic transformation?

• Single celled

• Use plasmid DNA

• Reproduce quickly

Source of “glowing gene” for this experiment

Aequorea victoria: jellyfish

Gene isolated by restriction enzymes and placed into plasmid

pGLOori

blaGFP

araC

pGLO Plasmid – 3 genes– Beta Lactamase

• Ampicillin resistance

– Green Fluorescent Protein

• jellyfish gene – glows

green in prescence of

arabinose sugar

– araC regulator protein

• Control gene (switches on in the presence of arabinose)

• Creates a protein that turns on GFP gene!

Arabinose Operon

RNA Polymerase

B A DaraC

Effector (Arabinose)

araC B A D

Promotor

(PBAD)

DNA binding Protein: Represses Transcription

Genes coding for digestive enzymes

B A DaraC

Transcription

Ara-GFP Operon

RNA Polymerase

araC GFP Gene

araC GFP Gene

araC GFP Gene

Effector (Arabinose)

Promotor (PBAD)

Transcription

GFP only produced in the GFP only produced in the presence of Arabinosepresence of Arabinose

Genes coding for Genes coding for digestive enzymes have digestive enzymes have been replaced by the GFP been replaced by the GFP gene: no metabolism of gene: no metabolism of arabinosearabinose

Bacterial TransformationPlasmids enter bacterial cell and

genes are expressed

GFP

pGLO plasmids

Beta lactamase(ampicillin resistance)

Bacterial chromosomal DNA

Cell wall

What is in the agar?

• LB – nutrient broth for bacteria to feed on

• Ampicillin – antibiotic that kills bacteria

• Arabinose – Sugar necessary to switch on ara C gene and the GFP gene

Transformation Procedure: Overview

• Suspend bacterial colonies in

Transformation Solution

• Add pGLO plasmid DNA

• Place tubes on ice

• Heat shock at 42oC and place on ice

• Incubate with LB nutrient broth

• Streak plates

Reasons for EachTransformation Step:

1.CaCl2 treatment (TS)Positive charge of Ca 2+

neutralizes:• negative charge of DNA • negative charge of cell membrane

2.Incubation on ice slows cell membranes3. Heat-shock

Increases permeability of cell membrane

These steps allow the plasmid to be taken in by the bacteria cells

4. Nutrient broth incubationallows beta lactamase gene to be expressed (for antibiotic resistance)

How will we know if bacteria is transformed?

• If bacteria grows in the presence of ampicillin

• If bacteria glows in the presence of arabinose

pGLO Lab

• Purpose: To transform E. coli bacteria by adding plasmids that allow the bacteria to glow green under UV light in the presence of arabinose sugar and grow in the presence of the antibiotic, ampicillin.

Sterile Technique

• Bacteria are UBIQUITOUS…they are found EVERYWHERE!

• Sterile technique refers to procedures that reduce the possibility of contamination…these techniques protect YOU, your CULTURES and REAGENTS, and LAB EQUIPMENT