Topic 4: Genetics - scienceystuff

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11 Δεκ 2012 (πριν από 4 χρόνια και 4 μήνες)

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IB Standard level Biology Dulwich College Shanghai

Topic 4: Genetics


4.4

Genetic Engineering and Biotechnology


Transgenesis

4.4.8

Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium,
yeast or other cell), restriction enzyme
s (endonucleases) and DNA ligase.


Ref:

Orange book


pg. 161

Green book


pg. 68
-
69



Introduction
-

**Do NOT learn

Since the discovery of the hormone insulin in 1921, diabetic patients, whose elevated sugar levels are due to
impaired insulin production,
have been treated with insulin derived from the pancreas glands of abattoir
animals. The hormone, produced and secreted by the beta cells of the pancreas' islets of Langerhans,
regulates the use and storage of food, particularly carbohydrates


Although bov
ine (cow) and porcine (pig) insulin are similar to human insulin, their composition is slightly
different. Consequently, a number of patients' immune systems produce antibodies against it, neutralising its
actions and resulting in inflammatory responses a
t injection sites. Added to these adverse effects of bovine
and porcine insulin, were fears of long term complications ensuing from the regular injection of a foreign
substance,

as well as a projected decline in the production of animal derived insulin. Th
ese factors led
researchers to consider synthesising
Human insulin

by inserting the insulin gene into a suitable vector, the
E.
coli

bacterial cell, to produce insulin that is chemically identical to its naturally produced counterpart. This
has been achie
ved using Recombinant DNA technology. This method is a more reliable and sustainable

method
than extracting and purifying the abattoir by
-
product.


1.

Identifying the human insulin gene


DO NOT LEARN

The human insulin gene was isolated, cloned and sequenced
in the 1970s, and so it became possible to insert
this gene into bacteria, who could then produce human insulin in large amounts.

We know which gene codes for insulin.


2.

Inserting the DNA into a plasmid vector using restriction enzymes and DNA ligase


Restr
iction Enzymes

These are enzymes that cut DNA at specific sites. They are properly called restriction endonucleases
because they cut
phosphodiester

(Between sugar and phosphate)

bonds in the middle of the polynucleotide
chain. Some restriction enzymes cut

straight across both chains, forming blunt ends, but most enzymes make
a staggered cut in the two strands, forming ‘sticky ends’.


The cut ends are “sticky” because they have short stretches of single
-
stranded DNA with complementary
sequences. These sti
cky ends will stick (or anneal) to another piece of DNA by complementary base pairing,
but only if they have both been cut with the same restriction enzyme. Restriction enzymes are highly
specific, and will only cut DNA at specific base sequences, 4
-
8 bas
e pairs long, called recognition sequences.





DNA Ligase

This enzyme repairs broken DNA by joining two nucleotides in a DNA
strand. It is commonly used in genetic engineering to do the reverse
of a restriction enzyme, i.e. to join together complementar
y
restriction fragments.




The sticky ends allow two complementary restriction fragments to
anneal, but only by weak hydrogen bonds, which can quite easily be
broken, say by gentle heating. The backbone is still incomplete.





DNA ligase completes the DN
A backbone by forming covalent
phosphodiester bonds. Restriction enzymes and DNA ligase can
therefore be used together to join lengths of DNA from different
sources.


Vectors

In biology
a vector is something that carries things between species
. For examp
le the mosquito is a disease
vector because it carries the malaria parasite into humans. In genetic engineering a vector is a length of
DNA that carries the gene we want into a host cell. A vector is needed because a length of DNA containing a
gene on its

own won’t actually do anything inside a host cell. Since it is not part of the cell’s normal genome it
won’t be replicated when the cell divides, it won’t be expressed, and in fact it will probably be broken down
pretty quickly. A vector gets round these

problems by having these properties:




It is big enough to hold the gene we want.



It is circular (or more accurately a closed loop), so that it is less likely to be broken down (particularly in
prokaryotic cells where DNA is always circular).



It contains c
ontrol sequences, such as a replication origin and a transcription promoter, so that the gene
will be replicated, expressed, or incorporated into the cell’s normal genome.



It contains marker genes, so that cells containing the vector can be identified.


Pl
asmids

Plasmids are by far the most common kind of vector, so we shall look at how they are used in some detail.
Plasmids are short circular bits of DNA found naturally in bacterial cells. A typical plasmid contains 3
-
5 genes
and there are usually around
10 copies of a plasmid in a bacterial cell. Plasmids are copied separately from
the main bacterial DNA when the cell divides, so the plasmid genes are passed on to all daughter cells. They
are also used naturally for exchange of genes between bacterial ce
lls (the nearest they get to sex), so
bacterial cells will readily take up a plasmid. Because they are so small, they are easy to handle in a test
tube, and foreign genes can quite easily be incorporated into them using restriction enzymes and DNA ligase.










The diagram below shows how DNA fragments can be incorporated into a plasmid using restriction and ligase
enzymes.


The foreign DNA anneals with the plasmid and is joined covalently by DNA ligase to form a hybrid vector (in
other words a mixtur
e or hybrid of bacterial and foreign DNA).


Make sure you understand this diagram





3.

Inserting the plasmid vector into the host bacterium

The plasmid must now be introduced into a bacterial cell, which will allow the vector to multiply (clone itself
and

the foreign donor DNA it contains). The bacterium commonly used is
Escherichia coli
(
E. coli
)

a normal
inhabitant of the human gut and was chosen for this task because a great deal is known about its genetics and
because it grows rapidly with a doubling
time of 30 minutes. A mutant form of
E. coli

was specially developed
for genetic engineering. This form can only survive in special laboratory conditions. Therefore if it escapes
with foreign genes inserted, it cannot infect humans.


Don’t memorise the
method

If a plasmid vector is being used, it is added to a flask containing a culture of
E. coli
. Calcium ions, usually in
the form of calcium chloride, are added to the flask, followed by a brief heat shock. This has the effect of
making holes appear br
iefly in the cell surface membrane of the
E. coli
, making them permeable to DNA and
allowing plasmids to enter. The process of adding new DNA to a bacterial cell is called
transformation
.


4.

Cloning the bacteria and harvesting the human insulin

The bacteria
l cells need nutrients in order to grow, divide, and live. While they live, the bacterial cell
processes turn on the gene for human insulin and the insulin is produced in the cell. When the bacterial cells
reproduce by dividing, the human insulin gene is

also reproduced in the newly created cells.


Human insulin protein molecules produced by bacteria are gathered and purified.

Millions of people with diabetes now take human insulin produced by bacteria or yeast (biosynthetic insulin)
that is genetical
ly compatible with their bodies, just like the perfect insulin produced naturally in your body.

Question One











(8)


Recombinant DNA technique (transgenesis) has enabled researchers to insert DNA from one organism into
the gen
ome of bacteria for later multiplication.


The numbered diagrams on the following page show the sequence of events in the process of gene cloning.


(a)

Match the numbers of the diagram with the descriptions below. (The first answer has been done for
you).







(4)

A.

A selected restriction enzyme cuts the donor DNA and the bacterial DNA do that they that the
same sticky ends. = 3

B.

The plasmids with the recombinant DNA are reincorpor
ated into bacteria.

C.

The donor DNA is joined by a ligase to produce recombinant DNA molecules.

D.

Recombinant bacteria are selected out to produce large numbers of recombinant plasmids.

E.

Donor cell DNA

F.

The restriction enzyme is used again to cut the segments of

donor DNA from the plasmids.

G.

Bacterial cell DNA.

H.

Donor DNA is incorporated into the bacterial plasmid.

I.

Recombinant DNA plasmids multiply inside the bacteria.




(b)

Refer to the following and give the numbers of the two ‘sticky ends’ resulting from a cut by t
he same
restriction enzyme.










(2)


1. GAA





2. GCCTGGC




3. AAGCCT



CTTAAG




ACCG




TTC



4. CTGCAG





5.

GAA




6. AAGCTT



GAC






GGACTT





GAA


(c)

State TW
O reasons why an experimenter might want to insert some donor DNA into a bacterial
plasmid.







(2)



Question Two














(14)


Read the following extract carefully,
then answer the questions which follow.


Volunteers are being recruited to eat raw potatoes in the first human trials of a vaccine
grown in genetically engineered potatoes. Researchers hope that people who eat the
potatoes will be protected from common gu
t infections.


The team first tried out the technique in tobacco plants. They took a strain of
Escherichia
coli

bacteria that causes food poisoning and identified that toxin as a protein molecule.
The toxin binds to receptor molecules on the cell surface

membrane of the gut cells of its
victim. The researchers then used a modified bacterium called
Agrobacterium
tumefaciens
. Under normal circumstances, these bacteria transfer plasmids into plant
cells causing the plant to manufacture the nutrients the ba
cteria need. In the modified
bacteria, however, the gene for producing the
E. coli

toxin had been inserted into the
plasmid DNA.


Once inside the tobacco cells, the foreign DNA becomes incorporated into the tobacco
chromosomes. These genetically engineer
ed tobacco plants were grown and propagated
asexually by taking cuttings. These cuttings all contained the gene for, and produced, the
E. coli

toxin.


Proof of success came when the tobacco leaves were mashed up and fed to mice. Within
days the mice star
ted producing antibodies specific to the
E. coli
toxin. The team then
produced genetically engineered potatoes and fed these to mice with similar results.



One problem with growing potatoes to produce vaccines is that the potatoes are usually
cooked befo
re being eaten. Bananas, which are usually eaten raw, might prove to be a
better option.







(a)

Use the information in this passage to help explain what is meant by:


(i)

recombinant DNA








(2)


(ii)

a vector









(2)


(b)

Explain why:


(i)

the tobacco cuttings all contained the gene for producing the toxin.




(2)


(ii)

Only some of the plants grown from the seeds of the genetically engineered tobacco plants would
be

expected to contain the gene.









(2)


(c)

Explain why it is thought that bananas might be a better option than potatoes for producing the vaccine.


(2)


(d)

What is a
plasmid
?













(1)


(e)

The gene for the
toxin may be isolated and put into a plasmid from
Agrobacterium tumefaciens
.
Describe in detail the part played by restriction enzymes in these processes.




(3)