pGLO Transformation
Transforming Genes
• Genetic transformation is a method of
inserting a gene into an organism in order to
change the trait(s) of the organism.
• This is used in agriculture (genes for frost
resistance etc), Bioremediation (bacteria used
to digest oil spills) and Gene Therapy (a
disease causing gene replaced with healthy
one).
The GFP gene.
• You will be transforming bacteria with a gene
that codes for ‘’Green Fluorescent Protein’’
• Real life source of this protein is the
Bioluminescent Jellyfish ‘’Aequorea Victoria’’
• http://www.youtube.com/watch?v=jCKKxi_bh
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Bioluminescence
• Other organisms in nature display
bioluminescence:
• http://www.youtube.com/watch?v=ku5OsM9
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GFP gene
• Scientists have successfully transformed the
GFP gene into a variety of organisms:
• http://www.youtube.com/watch?v=NfEIp2UJC
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• http://www.youtube.com/watch?v=fYN1KzFdj
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GFP
• At the end of this experiment, the bacteria will
express their newly acquired GFP gene and
will glow brilliant green under UV light.
• The gene will be transformed into the bacteria
with the use of a ‘’plasmid’’.
Plasmids
• Bacteria have DNA contained in one
chromosome.
• They also have small pieces of
Circular DNA called plasmids.
• Plasmids contain genes which are beneficial to
the bacteria’s survival.
• In nature, different bacteria can exchange
plasmids back and forth in order to ‘’share’’
beneficial traits.
Bio-Rad’s pGLO plasmid.
• This plasmid encodes for the GFP gene and a
gene for ampicillin resistance.
• It also contains a gene regulation system.
The Gene Regulation System
• The Arabinose Operon
• An operon is a set of genes that work
together.
• The arabinose operon uses both positive and
negative control.
The Arabinose Operon
• The operon contains three different genes (ara A, ara B, and
ara D) that code for three enzymes needed to convert
arabinose to a usable form. A fourth gene codes for a protein
(ara C) that acts to regulate the structural genes.
ara A, ara B, ara D
Regulatory
Gene
Activator
binding
site
Promoter
(Structural Genes)
The Arabinose Operon
Without Arabinose.
Ara C (=protein) binds to activator binding site.
This causes transcription of structural genes (ara A,B,D) = positive control
It also prevents transcription of ara C (as it is no longer needed.) = negative
control.
Ara C
ara A, ara B, ara D
Regulatory
Gene
Activator
binding
site
Promoter
(Structural Genes)
When Ara C binds to other sites, it inhibits the structural genes. These
genes are not usually active.
The Arabinose Operon
With Arabinose.
binds with the arabinose and changes shape
this promotes the attachment of RNA polymerase, leading to
transcription
Arabinose
Transcription
Ara C
ara A, ara B, ara D
Regulatory
Gene
Activator
binding
site
Promoter
(Structural Genes)
Bacteria
• Bacteria are used in this genetic transformation because:
• They are single celled. (so accessing the DNA is easier)
• They reproduce very quickly so the results of passing on genes
is seen very quickly.
Step 1: Observing E-Coli Colonies
In order to make observations later on about whether the
genetic transformations have taken place, the first stage is to
observe ‘normal’ e-coli colonies.
Consider the following:
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•
•
•
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Number of colonies
Size of: 1) the largest colony
2) The smallest colony
3) The majority of colonies
Colour of the colonies
Distribution of the colonies on the plate
Visible appearance when viewed with ultraviolet
(UV) light
The ability of the cells to live and reproduce in the
presence of an antibiotic such as ampicillin
Introducing the pGLO plasmid into E-Coli
cells.
• To move the pGLO plasmid DNA through the cell
membrane you will:
• 1. Use a transformation solution of CaCl2
(calcium chloride)
• 2. Carry out a procedure referred to as heat shock
• For transformed cells to grow in the presence of
ampicillin you must:
• 3. Provide them with nutrients and a short
incubation period to begin expressing their
newly acquired genes
Now carry out the procedure
• Follow the stages in the booklet
• When you have completed the practical, fill in
the answers to the questions on page 10.