Download Genetic Engineering and Gene Technology

Genetic Engineering and Gene Technology
define the term recombinant DNA;
explain that genetic engineering involves the extraction of genes from one organism, or
the manufacture of genes, in order to place them in another organism (often of a
different species) such that the receiving organism expresses the gene product
describe how sections of DNA containing a desired gene can be extracted from a donor
organism using restriction enzymes;
explain how isolated DNA fragments can be placed in plasmids, with reference to the role
of ligase;
state other vectors into which fragments of DNA may be incorporated;
explain how plasmids may be taken up by bacterial cells in order to produce a transgenic
microorganism that can express a desired gene product;
describe the advantage to microorganisms of the capacity to take up plasmid DNA from
the environment;
outline how genetic markers in plasmids can be used to identify the bacteria that have
taken up a recombinant plasmid;
outline the process involved in the genetic engineering of bacteria to produce human
insulin;
outline the process involved in the genetic engineering of ‘Golden Rice’
outline how animals can be genetically engineered for xenotransplantation
explain the term gene therapy;
explain the differences between somatic cell gene therapy and germ line cell gene
therapy;
discuss the ethical concerns raised by the genetic manipulation of animals (including
humans), plants and microorganisms
Genetic Engineering and Gene Technology: Key Terms
Recombinant DNA: A DNA molecule formed from DNA from 2 sources which are then joined
by the enzyme DNA ligase.
Genetic engineering: branch of biotechnology characterised by the obtaining of a specific
gene, placing the gene in a different organism and then causing the organism to express the
gene, transcribing and translating it to create a protein. This gives the recipient a
characteristic that it did not have previously.
Transgenic: an organism which contains DNA that has been added to its cells by genetic
engineering.
Transformed: a transgenic organism into which the new gene has been added is descried as a
transformed organism – for example, a bacteria that has taken up a DNA plasmid from the
environment.
Vector: a biological structure able to successfully introduce and integrate a gene into a cell
and its genome.
Annealing: the term used to describe hydrogen bond formation between complementary base
pairs when sections of DNA join together. Annealing is seen when complementary sticky ends
join.
DNA ligase: enzyme that catalyses the condensation reaction between the phosphate group
of one nucleotide and the sugar group of another to form the DNA sugar-phosphate backbone.
Reverse transcriptase: a retroviral enzyme that is able to construct DNA using an mRNA
strand as a template .
DNA polymerase: enzyme that synthesises new double stranded DNA – used to make the
single stranded cDNA produced by reverse transcriptase double stranded.
What is genetic engineering?
Genetic engineering is the term used to describe the processes by which a
specific gene is obtained, placed into another organism , causing the recipient to
express the new gene.
4 steps to genetic
engineering success:
1. Obtain the required
gene
2. Place a copy of the gene
in a vector
3. Allow the vector to
transfer the gene to the
target cell
4. Identify the cells that
Genetic engineering is used for 1 or 2 main reasons: to improve a
have successfully been
feature of the recipient organism or to engineer
organisms that can synthesis useful products. Inserting a
transformed. Allow
gene into crop plants to give resistance to herbicides will allow a
these to express the
farmer to use herbicides to kill weeds whilst the crop remains
new gene though protein unaffected. Inserting a gene for a human hormone such as insulin
synthesis.
into bacteria allows large quantities to be produced for human use.
Obtaining the gene
There are 3 ways to
do this...
1. Cut the gene out of the genome using
a restriction endonuclease enzyme
These enzymes cut through DNA at specific base sequence.
The sequence is known as the restriction site. Most restriction
enzymes catalyse a hydrolysis reaction which breaks the sugar
phosphate backbone of the DNA double helix in different places. They give a
staggered cut known as a ‘sticky end’ – a short run of unpaired exposed bases.
2. Extract the mRNA of a gene
from a cell which makes a lot of
that specific protein. Reverse
transcriptase enzyme can then be
used to convert the mRNA back
into a DNA copy of the gene. This is
used in the insulin production process
covered later in the power point.
3. If the gene
sequence is
known, the DNA
can be made in an
automated
polynucleotide
synthesiser.
What are vectors?
A vector is a
biological structure
able to successfully
introduce and
integrate a gene into
a cell and its genome.
Why do we use vectors?
1. To make sure that the gene is integrated into the genome and not
just deposited into the cytoplasm
2. Vectors contain genetic control regions to allow the cell to switch on
the gene and cause it to be transcribed and translated.
3. To ensure that the cell is undamaged by the insertion of extra DNA
There are 3 types of vectors that are commonly used in genetic
engineering: Bacteria Plasmids, Viruses and Liposomes.
A bacterial plasmid is a double stranded
circular DNA molecule that is separate from
the main chromosomal DNA.
Plasmids occur naturally in bacteria and
carry ‘extra, non essential’ genes that may
give a bacteria a survival advantage (like a
gene for antibiotic resistance).
Bacteria can exchange plasmids and take up
new DNA through a process called
conjugation. Bacteria reproduce asexually by
binary fission, so usually there is no genetic
variation. However, this exchange allows some
genetic variation to be introduced, and in
times of stress, such as in the presence of
antibiotics, the plasmids received may
increase chance of survival.
A viral vector ‘infects’ cells by binding to
specific complementary receptors on the
plasma membrane and then releasing their
viral DNA into the cytoplasm.
To use a virus as a vector, the required
gene is integrated into the virus and the
virus then integrates the gene as it infects
the cell.
Non pathogenic viruses are used, but
there is always a risk of mutation – the
virus could become pathogenic and cause
harm.
A liposome is a small sphere of lipid bilayer
containing a functioning allele of a gene. The
liposome passes through the phospholipid
bilayer and delivers the DNA into the
cytoplasm of the cell.
Putting the gene into the vector: making
recombinant DNA
If 2 pieces of DNA have been
cut with the same restriction
endonuclease enzyme, they
will have complementary
sticky ends. These unpaired
exposed bases can therefore
form hydrogen bonds by
complementary base pairing.
This is called annealing. The
enzyme DNA ligase then
catalyses condensation
reactions which joins the
sugar-phosphate backbone of
the DNA together to form
the recombinant DNA.
Cut with same
endonuclease restriction
enzyme = Complementary
sticky ends
Annealing and DNA
Ligase enzyme
Transforming the bacteria:
getting the recombinant plasmid into
the bacteria
Heat shock procedure
1.Mix the plasmids with the
bacteria population
2.Cool the mixture to near
freezing
3.Add a solution of calcium
salts
4.Quickly raise the
temperature to 40°
Usually, only about 1%
o the bacteria will
take up the plasmid
that contains the
desired gene.
Identifying the transformed bacteria
It is important to notice that not all the plasmids formed in this process will
contain the desired gene. We cut all of the DNA with the same restriction
endonuclease enzyme, so some plasmids will just reseal to form the original
plasmid once again. Three different types of bacteria can therefore be formed.
Some bacteria will have taken up no plasmid at all; some bacteria will have taken up
a plasmid that does not contain the desired gene and some bacteria (the ones that
we want!) will have taken up the recombinant plasmid – the transformed bacteria.
We need a way of identifying which are the bacteria with the recombinant plasmid.
The answer is to use plasmid vectors
with genetic markers. The original
plasmids are chosen because they
contain genes that make bacteria
resistant to 2 antibiotics – ampicillin
and tetracycline. The restriction
enzyme that is used to cut the plasmids
has its restriction site in the middle of
the tetracycline resistance gene. This
means that if the required gene is taken
up, then the tetracycline resistance
gene will be broken up and will not
function, but the ampicillin resistance
gene will still work.
This plasmid has
just resealed to
reform the
original. The
bacteria
containing it will
be resistant to
both ampicillin
and tetracycline.
This plasmid has taken up the required
gene, so the tetracycline gene is disrupted
and the bacteria containing it will not be
resistant to tetracycline.
Identifying the transformed bacteria:
Replica Plating
1. All of the
bacteria are grown
on nutrient agar.
All of the cells
grow to form
colonies.
2. A sterile cloth that
bacteria can stick to is
used to transfer some of
the cells from these
colonies to agar containing
the ampicillin antibiotic.
We therefore want the bacteria that were
alive on the ampicillin plate but dead on the
tetracycline plate. We can identify these by
comparing the plates and the harvest the
bacteria containing the recombinant plasmid
to produce the product on a large scale.
3. A sterile cloth that bacteria
can stick to is used to transfer
some of the cells from these
colonies to agar containing the
tetracycline antibiotic.
4. The bacteria that are
present on the agar that
contains ampicillin must have
a plasmid – all those that
have died are the bacteria
that did not take up a
plasmid at all. The bacteria
that survived on the
tetracycline plate have the
original plasmid with both
antibiotic resistance genes
in tact and therefore these
bacteria do not contain the
desired gene.
Case study 1: Human Insulin
People who cannot produce enough insulin suffer from type 1 diabetes. This is an autoimmune
condition in which the pancreatic Β-islet cells are destroyed. Before genetic engineering was
developed, diabetics were treated using pig insulin. There were some problems with this:
 Human insulin and pig insulin are not identical, so pig insulin isn’t as effective as human insulin.
Pig insulin can cause adverse reactions in patients
Pig insulin is very expensive to produce
Shortage of insulin because only a very small amount of insulin is present
in the pig’s pancreatic tissue
Ethical/moral objections to using animals
Genetic engineering now provides a solution – insulin can be mass
produced using bacteria!
Trying to find
the one insulin
gene in the
human genome
would be too
difficult, so
instead we use
reverse genetics
to get the gene
we want.
 Separate the cell’s mRNA from all other molecules
using basic centrifugation
 Use specialised centrifugation to isolate the mRNAs
which correspond to the insulin gene by length.
 We take mRNA that is present in large concentrations
in the B-Islet cells in the pancreas. We work
backwards from this, using the enzyme reverse
transcriptase, to go from single stranded mRNA to
double stranded DNA.
Making Human Insulin
1. Carry out reverse transcription:
take the mRNA of the insulin gene
and mix with reverse transcriptase
enzyme. Add DNA polymerase and
free DNA nucleotides to create
cDNA.
2. To allow the insulin gene to join with
the cut plasmid, they must have
complementary sticky ends. This
can be achieved by using the same
restriction enzyme to cut the cDNA
and the plasmid or by adding
artificial sticky ends to those of
the plasmid.
3. The vector is prepared by
cutting the plasmid with the same
restriction enzyme as was used
to cut the cDNA.
Making Human Insulin
4. The insulin DNA and plasmid are mixed
together with Ligase enzyme. The
complementary sticky ends anneal and
the Ligase enzyme joins the sugarphosphate backbone. A recombinant
plasmid is formed.
Insulin
DNA
Plasmid
5. Heat shock – recombinant plasmid introduced into bacteria
6. Identify the transformed bacteria using replica plating
7. The transformed bacteria containing the insulin gene can
produce insulin for human use!
Case Study 2: Golden Rice
Vitamin A deficiency can cause serious health
problems, including Xerophthalmia . Vitamin A
is needed for:
Maintaining vision at low light intensities
Effective immune system
Large numbers of people in areas such as
Africa and India, where the staple food is
rice, suffer from the effects of vitamin A
deficiency.
Genetic engineers
therefore found a
Vitamin A itself comes only from
way to add genes
animal sources, but it can be
to the rice plant
synthesised in the gut from beta
that would allow
carotene – a precursor. Beta
for the
carotene is a photosynthetic
transcription and
pigment, so it is only expressed in
translation of the
the parts of the rice plant that
beta-carotene in
photosynthesise – this is the hull
the endosperm
and bran layers. We only eat the
that is eaten by
endosperm, in which the beta
humans.
carotene genes are switched off.
Case Study 2: Golden Rice
Most of the enzymes needed for the synthesis of beta carotene were already present in
the endosperm. The insertion of 2 genes into the rice genome was needed in order for
the metabolic pathway to be activated in the cells of the endosperm.
Precursor
molecules
The genes codes for the
following enzymes:
From daffodils
Phytoene Synthetase
Phytoene
Crt1 enzyme
From bacteria
Lycoprene
Other Enzymes
The first version of golden rice
contained 4 x more beta
carotene than normal rice, but
this still meant that you’d have
to eat large amounts of rice
every day in order to prevent
vitamin A deficiency.
Beta
Carotene
Already present in
endosperm
Further genetic engineering added stronger promoters to the vitamin
A genes. These promoters increased the transcription of the betacarotene synthesising enzymes, so ‘Golden Rice 2’ contained over 20x
the original amount of beta carotene in the endosperm.