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genetic engineering
module 2 – biotechnology & gene technologies
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learning objectives

Understand what is meant by genetic engineering.

Understand the steps involved.

Understand what restriction enzymes do.

Understand why sticky ends are important.
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success criteria

State the definition of genetic engineering.

Describe what restriction enzymes do.

Explain the importance of sticky ends.
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From the spec
starter
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+ starter 2

Try to come up with a definition for the term – genetic
engineering.
The definition:
The transfer of genes from one organism to another (often a
different species). The organism receiving the gene expresses
the gene product through protein synthesis.
+ genetic engineering

Genetic engineering is a rapidly advancing field of Biology.

We can now manipulate, alter and even transfer genes
from one organism to another.

The ability to do these things has proved invaluable in the
industrial and medical sectors.
+ requires...
The following steps are necessary:
1.
The required gene is obtained.
2.
A copy of the gene is placed into a vector.
3.
The vector carries the gene to the recipient cell.
4.
The recipient expresses the gene through protein
synthesis.
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sticky ends
+ cutting the genes out

In order to isolate a gene, it needs to be cut from the donor
organisms DNA.

This is done using ‘molecular scissors’ known as restriction
enzymes.
Cuts made with restriction enzymes can have two results:
Some restriction
endonuclease
produce ‘blunt ends’
Some restriction
endonuclease
produce ‘sticky
ends’
+ importance of sticky ends

Restriction enzymes that cut the sugar-phosphate backbone
in different places, produce sticky ends.

These are really important due to the exposed bases left at
the staggered cut.

Due to the complimentary nature of DNA bases, sticky ends
on one gene, will pair up with sticky ends on another bit of
DNA, - provided it has also been cut with the same restriction
enzyme.
This gene (from a human) can be cut with
a restriction enzyme such as EcoRI
Sticky End
KEY:
Gene from Human
Gene from E.coli
This is a section of DNA from E.coli.
Sticky End
If this section of
DNA from E.coli
is also cut with
EcoRI, a
complimentary
sticky end is
produced.
If these two ‘cut’ pieces of DNA are mixed,
recombinant DNA has been produced.
Once the bases have paired, they form their
usual weak hydrogen bonds between each
other.
The only thing left to do, is form the link
between the sugar-phosphate backbones,
and this is done by the enzyme, DNA Ligase.
+ Questions
1.
Explain why different restriction enzymes have different
restriction sites (recognition sequences).
2.
Explain why restriction enzymes can be a useful defense
mechanism for bacteria against viruses.
3.
If bacterial DNA contains base sequences that are the same
as the restriction sites of their enzymes, these sites are
methylated (-CH3 group added). Explain why.
4.
The restriction enzyme EcoR1 was the first restriction
enzyme isolated from E. coli. Suggest how restriction
enzymes are named.
plenary
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Exam practice
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Answers
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learning objectives

Understand what is meant by genetic engineering.

Understand the steps involved.

Understand what restriction enzymes do.

Understand why sticky ends are important.
+
success criteria

State the definition of genetic engineering.

Describe what restriction enzymes do.

Explain the importance of sticky ends.
+
+
Insertion of DNA into a vector

VECTOR – used to transport DNA into a host cell.

PLASMID – the most commonly used vector. A circular piece
of DNA found in bacteria.

Plasmids are useful because the nearly always contain
antibiotic resistance genes (see later).
The Plasmid
 One
of the antibiotic
resistant genes is
disrupted when the
restriction enzymes
cuts open the
plasmid.
 The
other antibiotic
resistant gene is used
in selection of the
correct host cells.
(See later)
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Insertion into plasmids

What combinations of plasmid will form?
Inserting genes into Plasmids
• The real-life application of what we have just learnt, occurs
when geneticists insert an animal or plant gene into plasmids.
This
‘Step
2’ (insertion)
the
process
• Plasmids
areissmall
loops
of DNA whichinare
found
in addition
to the large of
circular
chromosome
that
bacterial
cells possess.
making
a protein
using
gene
• By inserting our chosen technology
gene into a plasmid, the plasmid acts
as a ‘carrier’, or vector, which we can then introduce back into
a bacterial cell.
Restriction Endonuclease
Restriction Endonuclease
DNA coding for a desired
protein
Remember, that DNA
Ligase would once again be
used to bond the sugarphosphate backbones.
A plasmid
As the DNA fragment was cut out using
the same restriction endonuclease as
used to cut the plasmid open, they have
complimentary sticky ends.
Discussion questions
• Why was it important to find an enzyme that
would cut once in the plasmid?
• What other considerations were there in
choosing the enzyme to cut the plasmid and
DNA sequence (think shaded areas).
• How can we use the new recombinant
plasmid to produce insulin?
Introducing our recombinant plasmids into host cells
• Introducing recombinant plasmids into bacterial cells is called
transformation.
• This is done by mixing the plasmids with the cells in a
medium containing calcium ions, and changing the
temperature
• The calcium ions make the bacterial cells permeable, allowing
the plasmids to pass through, into the cell.
However, only a few bacterial cells
(approx 1%) will actually take up
the plasmids.
Calcium ion medium
For this reason, we need to
identify which ones have been
successful. This is done with gene
markers.
This is ‘Step 3’ (transformation) of producing a
protein by DNA technology
plasmid
Insulin
Bacterial chromosome
Using Gene Markers to identify successful host cells...
• There are a number of different ways of using gene markers to
identify whether a gene has been taken up by bacterial cells.
• They all involve using a second, separate gene on the plasmid. This
second gene acts as a ‘marker’ because....
• It may give resistance to an antibiotic
• It may make a fluorescent protein that is easily seen
• It may produce an enzyme whose action can be identified
1. Antibiotic-Resistance Markers
• Many bacteria contain antibiotic resistance genes in their plasmids.
Some in fact, can have two genes for resistance to two different
antibiotics, in the same plasmid.
Gene for
resistance to
tetracycline
Gene for
resistance to
ampicillin
Any bacterial cell possessing this
plasmid, would be resistant to both of
the antibiotics, ampicillin and
tetracycline.
But what if we cut right in the middle
of the tetracycline-resistance gene
(with a restriction endonuclease), and
insert a gene of our own interest?
Bacteria with this plasmid
would only be resistant to
ampicillin, not
tetracycline.
How is this of any
advantage to us?
First, the recombinant plasmids are
introduced into bacterial host cells
(transformation)
The bacteria are grown on agar
plates treated with ampicillin
Colonies are
allowed to grow,
but will only do so
if they are resistant
to ampicillin – i.e.
Bacteria that took
up the plasmid.
A replica plate is now made. This is when you literally press the agar of one
Petri-dish, onto the agar of a new Petri-dish, transferring bacterial cells from
each colony onto the new agar.
This agar
however,
has been
treated
with
tetracycline
?
Colonies are allowed to
develop
There is a missing
colony, which has
lost resistance to
tetracycline.
This must be a
colony of cells
which have taken
up the
recombinant
plasmid!
2. Fluorescent Markers
• This is a more recent method of finding out whether bacteria have taken up
the desired plasmids.
Throughout nature, there are organisms such as
jellyfish, that produce fluorescent proteins.
These proteins, coded for by their own genes, can
be isolated and then introduced into bacterial cells
via vectors.
The range of natural fluorescent proteins can be
seen on this Petri-dish.
• First the fluorescence gene is inserted into a plasmid vector
• Using restriction enzymes, the gene of interest (e.g. Human insulin gene) is
then inserted into the middle of the fluorescence gene, so the latter can no
longer be expressed
• Bacteria that have taken up the plasmid alone will fluoresce under a
microscope BUT those containing the recombinant plasmid will not fluoresce
• This is an easier way to identify bacteria expressing the gene of interest
3. Enzyme Markers
• This method involves inserting your gene of interest (e.g. Insulin),
into a gene that codes for an enzyme such as lactase.
• There is a particular substrate that is usually colourless, but turns
blue when lactase acts upon it.
• If you insert your chosen gene into the gene that makes lactase,
you will inactivate the lactase gene.
• If you now grow bacterial cells on an agar medium containing the
colourless substrate, any bacteria that have taken up the
recombinant plasmid, will form white colonies not blue ones.
Gene Therapy
• What are some of the ethical considerations for using gene therapy?
• --Some Questions to Consider...
– What is normal and what is a disability or disorder, and who decides?
– Are disabilities diseases? Do they need to be cured or prevented?
– Does searching for a cure demean the lives of individuals presently
affected by disabilities?
– Is somatic gene therapy more or less ethical than germline gene
therapy?
– In cases of somatic gene therapy, the procedure may have to be
repeated in future generations.
– Preliminary attempts at gene therapy are exorbitantly expensive. Who
will have access to these therapies? Who will pay for their use?
• Cystic fibrosis is the most common inherited
disease in the UK.
• It affects 1 in every 2500 people.
1 in 25 people carry the faulty gene that causes
cystic fibrosis.
What is gene therapy?
• Gene therapy replaces defective genes
with healthy ones to treat genetically
inherited diseases.
• The cause of the disease is tackled not
just the symptoms.
• Cystic fibrosis is an inherited disease
that can be treated by gene therapy.
• Gene therapy has been used in the
treatment of cancer and recently, in
2008 to treat genetic blindness
Cystic fibrosis
 Cystic fibrosis is a recessive genetic disease which affects 1 in
2500 people in the UK. 1 in 20 are carriers!
 Symptoms include:
◦ Production of thick sticky mucus from the cells that line
respiratory system
◦ causes congestion of the lungs, breathing difficulties and
higher risk of infection.
◦ Blockage of the pancreatic duct with mucus
◦ can lead to a reduced release of pancreatic juice and
inadequate digestion.
◦ prevents pancreatic enzymes from reaching the duodenum and
leading to the formation of fibrous cysts.
◦ The reproductive system is also blocked, preventing
sufferers having children.
Treatment using gene therapy
There are 2 ways in which gene therapy can
be used to treat cystic fibrosis
1) Gene replacement – defective genes are
replaced by healthy ones
2) Gene supplementation – one or more
copies of the healthy gene are added
alongside the defective gene.
3) The added genes are dominant so they
mask the effects of the recessive alleles.
Gene therapy techniques
1) Germ-line therapy – replacing/supplementing the defective gene
in the fertilised egg. All cells will develop normally and the cells of
the offspring
2)Somatic-cell therapy – targets the affected tissues, such as the
lungs,
. Long term is to target undifferentiated stem cells so treatment is
effective for the life span of the individual.
Name at least one issue with each type of therapy
1) Moral and ethical issues
2) Treatment needs to be repeated – limited success
How can somatic-cell gene therapy
help?
It involves inserting cloned functional CFTR
genes into epithelial cells of the lungs to
replace defective genes.
2 techniques are being tested:
(1) Wrapping CFTR genes in lipids that can
be absorbed through the cell surface
membranes.
(2) Inserting CFTR genes into harmless
viruses called adenoviruses that are then
allowed to ‘infect’ the cells.