Transcript Transformation and Protein Purification

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
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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


bla is an ampicillin
resistance gene
ori
bla
Bacterial Cells and DNA
Chromosomal
Bacterial cell
Plasmid DNA
Chromosomal DNA
Growth of Bacteria
on Plates
bacteria
If few
cells grow
colonies
Agarose in Petri
dish = plate
Incubate
at 37C
If many
cells grow
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
DNA after heat shock
cells uptake
The pGLO Plasmid
pGLO Plasmid
 bla
gene
 beta-lactamase

enzyme
araC
Ampicillin resistance
 GFP
gene
ori
Fluorescent Protein
 Aequorea victoria jellyfish
pGLO
 Green
 araC
gene
 Regulates
GFP transcription
ori
Allows plasmid replication
bla
GFP
pGLO Plasmid: Most
Important Components

bla gene
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Bacteria with this gene grow
in the presence of ampicillin
pGLO
GFP gene

Bacteria with this gene glow
under near UV light
bla
GFP
Experimental Procedures
Transformation Procedures
+CaCl2
+CaCl2
Transformation Procedures
Reasons for Each
Transformation Step
Ca++
CaCl2 treatment
O
Ca++
O P O
Base
O
CH2
Positive charge of Ca2+
ions neutralizes:
• negative charge of DNA
phosphates
• negative charge of
membrane phospholipids
O
Sugar
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
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
Transformation Results
Lb/amp/ara
Only cells
getting pGLO
plasmid grow
and glow
All cells grow
since there is
no antibiotic on
the plate
White – no glow
Without pGLO
plasmid,
nothing can
grow
All cells grow
since there is
no antibiotic
on the plate
Extension Activities
Extension Activity I:
Transcriptional Regulation
Arabinose controls expression of GFP gene:
Transfer Bacteria
Glowing Bacteria
from Transformation
Plate with
Arabinose
Incubate overnight @ 37C
Plate without
Arabinose
Extension Activity I:
Transcriptional Regulation
arabinose = no glow
+arabinose = glow
After overnight incubation
Plate with
Arabinose
Plate without
Arabinose
Transcriptional Regulation
of GFP by Arabinose
araC
GFP Gene
araC repressor blocks
transcription
Arabinose
araC
araC
GFP Gene
RNA Polymerase
GFP Gene
Arabinose binds repressor,
changing its conformation
Altered repressor leaves
DNA, RNA polymerase can
perform transcription
Extension Activity II:
Tweaking the Transformation Protocol
Test effect of various components of the
transformation protocol:
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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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