Transcript genetic engineering - Skinners` School Science

Genetic Engineering learning
outcomes
• 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
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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.
Restriction enzymes
(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
•
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.
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Engineering plasmids
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.
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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.
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Transformation in bacterial cells
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
•
•
State other vectors into which
fragments of DNA may be incorporated.
Eg: virus genomes or yeast cell
chromosomes
LO’s for Insulin and Golden Rice
• 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™.
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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.
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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
•
•
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•
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
Golden Rice
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