Transformation and Protein Purification

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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.
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Aequorea victoria Source of glowing gene for
this experiment
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Jellyfish Gene put into Other Critters
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Outline

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

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Overview
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What is Bacterial Transformation?

• Taking up of DNA from the environment by
  bacterial cells

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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

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Bacteria and Plasmids
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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
• bla is an ampicillin resistance gene

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Bacterial Cells and DNA
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Growth of Bacteria on Plates
Agarose in Petri dish plate
Incubate at 37?C
lawn
colonies
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Transformation
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Bacterial Transformation
The uptake of DNA
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Methods of transformation

• Electroporation
• Electrical shock makes cell membranes permeable
  to DNA
• Calcium Chloride/Heat Shock
• Chemically-competent cells uptake DNA after heat
  shock

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The pGLO Plasmid
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pGLO Plasmid

• bla gene
• beta-lactamase enzyme
• Ampicillin resistance
• GFP gene
• Green Fluorescent Protein
• Aequorea victoria jellyfish
• araC gene
• Regulates GFP transcription
• ori
• Allows plasmid replication

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pGLO Plasmid Most Important Components

• bla gene
• Bacteria with this gene grow in the presence of
  ampicillin
• GFP gene
• Bacteria with this gene glow under near UV light

pGLO
GFP
bla
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Experimental Procedures
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Transformation Procedures
CaCl2
CaCl2
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Transformation Procedures
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Reasons for Each Transformation Step

• CaCl2 treatment
• Positive charge of Ca2 ions neutralizes
• negative charge of DNA phosphates
• negative charge of membrane phospholipids

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Reasons for Each Transformation Step

• Incubation on ice slows fluid cell membranes
• Heat-shock increases permeability of cell
  membrane
• Nutrient broth incubation allows beta lactamase
  expression

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Transformation Results
Lb/amp/ara
Only cells getting pGLO plasmid grow and glow
All cells grow since there is no antibiotic on
the plate
Without pGLO plasmid, nothing can grow
All cells grow since there is no antibiotic on
the plate
White no glow
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Extension Activities
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Extension Activity I Transcriptional Regulation

• Arabinose controls expression of GFP gene

Incubate overnight _at_ 37?C
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Extension Activity I Transcriptional Regulation

• ?arabinose no glow
• arabinose glow

After overnight incubation
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Transcriptional Regulation of GFP by Arabinose
araC repressor blocks transcription
Arabinose binds repressor, changing its
conformation
Altered repressor leaves DNA, RNA polymerase can
perform transcription
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Extension Activity II Tweaking the
Transformation Protocol

• Test effect of various components of the
  transformation protocol
• plate ampicillin concentration
• plate arabinose concentration
• amount of plasmid DNA used in the experiment
• amount of cells used in the experiment
• length of time cells/DNA mix is kept at 42?C
  during the experiment

Compare results with number of colonies obtained
during the normal protocol
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