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

pGLO ori bla GFP 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 BAD araC Effector (Arabinose) araC BAD Promotor (P BAD ) DNA binding Protein: Represses Transcription Genes coding for digestive enzymes BAD araC Transcription

Ara-GFP Operon RNA Polymerase araC GFP Gene araC GFP Gene araC GFP Gene Effector (Arabinose) Promotor (P BAD ) Transcription  GFP only produced in the presence of Arabinose  Genes coding for digestive enzymes have been replaced by the GFP gene: no metabolism of arabinose

Bacterial Transformation Plasmids 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 42 o C and place on ice Incubate with LB nutrient broth Streak plates

Reasons for Each Transformation Step: 1.CaCl 2 treatment (TS) Positive charge of Ca 2+ neutralizes: negative charge of DNA negative charge of cell membrane

2.Incubation on ice slows cell membranes 3. Heat-shock Increases permeability of cell membrane These steps allow the plasmid to be taken in by the bacteria cells

4. Nutrient broth incubation allows 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