Genetic Engineering - Skinners' School Science

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

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© Pearson Education Ltd 2009

This document may have been altered from the original



Define the term
recombinant

DNA
.



A section of DNA (often in the form of a
plasmid
) which
is formed by
joining DNA sections from 2 different
sources


Genetic

Engineering

learning
outcomes

Genetic

Engineering

learning
outcomes


Be able to explain that
genetic
engineering

involves the
extraction of genes from one organism and placing
them into another organism.



Be able to describe how sections of DNA containing a
desired gene can be extracted from a donor organism
using
restriction enzymes.




Be able to explain how isolated DNA fragments can be
placed in plasmids, with reference to the role of
ligase
.



(a)

Cutting DNA using a
restriction enzyme
produces a staggered
cut and leaves
sticky
ends.
They cut at
specific sequences in the
DNA called restriction
sites
(or recognition sites)


(b)

Joining DNA from two
sources cut with the
same restriction enzyme;
complementary sticky
ends allow the base
pairs to
anneal
or match
up

and form H bonds


Make sure you can
explain the naming of
restriction enzymes

Restriction enzymes

© Pearson Education Ltd 2009

This document may have been altered from the original



Explain how engineered 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.

© Pearson Education Ltd 2009

This document may have been altered from the original


Bacterial cells can be
encouraged to take up
plasmids (circular pieces of
DNA found in bacteria)
containing foreign genes
by
treating them with
calcium
salts.


The cells receiving the
plasmids are
transgenic.


Transgenic organisms
contain
additional DNA which has
come from another organism


The transgenic bacteria can
be cultured and will express
the inserted genes as if they
were their own.

Engineering plasmids

© Pearson Education Ltd 2009

This document may have been altered from the original


Bacterial conjugation

Bacteria often transfer plasmids
from one to another. Because the
plasmids often carry genes for
antibiotic resistance this speeds
up the spread of this
characteristic in bacterial
populations, leading to strains
such as MRSA.


This is an advantage to the
bacteria but creates problems for
the medical profession.

Bacteria are injected into mice


S strain of bacterium contains a gene to
make a toxic protein but R strain of
bacterium does not.



Heat killed S strain bacteria can’t make
the protein so the mouse remains
healthy.



R strain mixed with killed S strain kills
the mouse because the live bacteria
can take up the DNA from the dead
bacteria and use its “instructions” to
make the toxic protein.

This is called
TRANSFORMATION





Transformation in bacterial cells


State other vectors into which
fragments of DNA may be incorporated.


Eg: virus genomes or yeast cell
chromosomes


© Pearson Education Ltd 2009

This document may have been altered from the original



Outline the process involved in the genetic engineering of
bacteria to produce human insulin.



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 genetic engineering
Golden Rice™.


LO’s for Insulin and Golden Rice

© Pearson Education Ltd 2009

This document may have been altered from the original


Production of bacteria containing the human
insulin gene


mRNA for insulin extracted from pancreatic cells
by differential centrifugation, 153 base pairs
corresponds to 51 amino acids.


Use Reverse Transcriptase enzyme to produce
single strand of DNA


Then use DNA polymerase and free nucleotides
to produce complementary strand.


Double strand cDNA (copy DNA) is the gene for
insulin


Sticky ends added, sometime promoter site is
added


Cut plasmid with restriction enzyme to give
complementary sticky ends


Add cDNA and plasmid together and use ligase to
join the sugar phosphate backbones.


Add the recombinant plasmids to bacteria with
calcium salts


Some bacteria will take up plasmids, some
plasmids will have taken up the insulin gene.


Identifying Transformed Bacteria


By using plasmids that carry genes for
resistance to ampicillin and tetracycline it
is possible to identify those bacteria that
have taken up plasmids that contain the
desired gene.



The desired gene is inserted part way
along the tetracycline resistance gene.



Bacteria are grown on ampicillin agar so
all bacteria that have plasmids in them
grow



Replica plating is carried out on
tetracycline agar


only those bacteria
that HAVEN’T taken up the insulin gene
will grow on this.



So it is possible to identify those colonies
on the ampicillin plates that DO have the
insulin gene.

Golden Rice


500,000 affected by
Vit

A blindness annually


1
-
2 million deaths


Vit

A found only in animal sources


Beta carotene is a plant
-
made precursor to
Vit

A
converted to
vit

A in gut


Needed for making
rhodopsin
,
glycoproteins
,
epithelial cells, bone growth


Also need sufficient fat in diet as it is lipid soluble

Golden Rice


Rice plants have the gene for beta carotene (photosynthetic
pigment) but in the endosperm of rice it is not expressed


Need to add genes for “
Phytoene

synthetase
” from daffodils
and “
Crt

enzyme” from soil bacteria
Erwinia

uredovora

for
beta carotene to be made in endosperm


These 2 genes were inserted close to the promoter site that
initiates endosperm development so that they are switched
on at the same time.


Cross breeding of Golden Rice varieties has increased Beta
carotene production in endosperm by 20 fold


Need to eat about 200
-
300 grams of rice per day to get
sufficient
betacarotene



© Pearson Education Ltd 2009

This document may have been altered from the original


Golden Rice