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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 Hitomi
Monte Vista High School, Danville, CA.
Kirk Brown
Tracy High School, Tracy, CA.
Aequorea victoria:
Source of “glowing gene” for this experiment
Jellyfish Gene put into Other Critters
Outline
• Overview
• Bacteria and Plasmids
• Transformation
• The pGLO Plasmid
• Experimental Procedures
• Extension Activities
Overview
What is Bacterial
Transformation?

Taking up of DNA from the


environment by bacterial cells
Bacterial Transformation Lab

• Bacterial Cells and plasmid DNA


are mixed

• Cells take up plasmid

• Cell/DNA mix is plated on nutrient


agar with antibiotic

• Only cells which obtained plasmid


DNA will grow… and glow
Bacteria and Plasmids
What is a plasmid?

„ Small circular DNA molecule


„ Replicates autonomously
„ Originally evolved in bacteria
„ May contain antibiotic
resistance gene or be modified
to contain other genes ori

„ bla is an ampicillin bla


resistance gene
Bacterial Cells and DNA

Chromosomal

Bacterial cell

Plasmid DNA

Chromosomal DNA
Growth of Bacteria
on Plates
bacteria Agarose in Petri
dish = plate

Incubate
If few
at 37°C If many
cells grow cells grow

colonies lawn
Transformation
Bacterial Transformation
The uptake of DNA
Bacterial Cell
Chromosomal
DNA

Plasmids
Methods of transformation

„ Electroporation
„ Electrical
shock makes cell
membranes permeable to DNA

„ Calcium Chloride/Heat Shock


„ Chemically-competent cells uptake
DNA after heat shock
The pGLO Plasmid
pGLO Plasmid
„ bla gene
„ beta-lactamase enzyme
„ Ampicillin resistance araC

„ GFP gene ori


pGLO
„ GreenFluorescent Protein
„ Aequorea victoria jellyfish GFP
bla

„ araC gene
„ Regulates GFP transcription
„ori
„Allows plasmid replication
pGLO Plasmid: Most
Important Components

„ bla gene
„ Bacteria with this gene grow
in the presence of ampicillin pGLO

„ GFP gene GFP


bla
„ Bacteria with this gene glow
under near UV light
Experimental Procedures
Transformation Procedures

+CaCl2 +CaCl2
Transformation Procedures
Reasons for Each
Transformation Step
Ca++
O
nCaCl2 treatment Ca++ O P O
Base
O
CH2 O
Positive charge of Ca2+ Sugar
ions neutralizes:
• negative charge of DNA O
phosphates Ca++ O P O Base
• negative charge of O
membrane phospholipids CH2 O
Sugar

OH
Reasons for Each
Transformation Step

oIncubation on ice slows


fluid cell membranes

pHeat-shock increases
permeability of cell membrane

qNutrient broth incubation


allows beta lactamase expression
Biotechnology
Explorer Program

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