LAB 6 pGlo Powerpoint - Bremen High School District 228
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Transcript LAB 6 pGlo Powerpoint - Bremen High School District 228
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 37C
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
Ampicillin resistance
GFP
gene
Green
Fluorescent Protein
Aequorea victoria jellyfish
araC
ori
pGLO
bla
gene
Regulates
araC
GFP transcription
ori
Allows plasmid replication
GFP
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
bla
GFP
pGlo Plasmid Additional Features
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1. Arabinose
sugar binds
regulator.
2. Regulator plus
arabinose complex
recruits RNA
Polymerase to
bind promotor.
3. Gene is
transcribed into
mRNA and “read.”
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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 @ 37C
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:
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 42C during
the experiment
Compare results with number of colonies
obtained during the normal protocol
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