This Little Light of Mine:This Little Light of Mine:Transform bacteria with a Jellyfish

gene to make them glow

Module based on a kit from Bio-Rad Laboratories, Inc.

Adapted by Dan Murray from a presentation by

Stan HitomiMonte Vista High School, Danville, CA.

Kirk BrownTracy High School, Tracy, CA.

Aequorea victoria: Source of “glowing gene” for this experiment

Jellyfish Gene put into Other CrittersJellyfish Gene put into Other Critters

OutlineOutline

• Overview • Bacteria and Plasmids• Transformation• The pGLO Plasmid• Experimental Procedures• Extension Activities

OverviewOverview

What is Bacterial What is Bacterial Transformation?Transformation?

Taking up of DNA from the environment by bacterial cells

Bacterial Transformation LabBacterial Transformation Lab

• Only cells which obtained plasmid DNA will grow… and glow

• Cell/DNA mix is plated on nutrient agar with antibiotic

• Cells take up plasmid

• Bacterial Cells and plasmid DNA are mixed

Bacteria and PlasmidsBacteria and Plasmids

What is a plasmid?What is a plasmid?

Small circular DNA molecule Replicates autonomously Originally evolved in bacteriaMay contain antibiotic

resistance gene or be modified to contain other genes

bla is an ampicillin resistance gene

ori

bla

Bacterial Cells and DNABacterial Cells and DNA

Chromosomal DNA

Chromosomal

Bacterial cell

Plasmid DNA

Growth of Bacteria Growth of Bacteria on Plateson Plates

Agarose in Petri dish = plate

bacteria

Incubate at 37°CIf few

cells growIf many

cells grow

colonies lawn

TransformationTransformation

Bacterial Transformation

Plasmids

Chromosomal DNA

Bacterial Cell

The uptake of DNA

Methods of transformation

ElectroporationElectrical shock makes cell membranes permeable to DNA

Calcium Chloride/Heat ShockChemically-competent cells uptake DNA after heat shock

The pGLO PlasmidThe pGLO Plasmid

pGLOori

blaGFP

araC

pGLO Plasmid

bla genebeta-lactamase enzyme

Ampicillin resistance

GFP geneGreen Fluorescent ProteinAequorea victoria jellyfish

araC geneRegulates GFP transcription

oriAllows plasmid replication

pGLO

blaGFP

pGLO Plasmid: Most Important Components

bla geneBacteria with this gene grow in the presence of ampicillin

GFP geneBacteria with this gene glow under near UV light

Experimental ProceduresExperimental Procedures

Transformation Procedures

+CaCl2 +CaCl2

Transformation Procedures

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

PO

O

O

Base

OH

Sugar

Sugar

OCa++

Incubation on ice slows fluid cell membranes

Heat-shock increases permeability of cell membrane

Nutrient broth incubationallows beta lactamase expression

Reasons for Each Transformation Step

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