Agenda –
Applying DNA knowledge to diabetes
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Warm-up:
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Diabetes, Insulin, and rDNA
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Attractive & professional thank-you notes
Review
Recombinant DNA
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Plasmids
Recombinant Paper Plasmid
Recombinant DNA project
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Notes on what you have learned – due
tomorrow
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Electronic presentations
http://prezi.com/ezf2bged4bia/geneticengineering-and-biotechnology/Genetic
Engineering and Biotechnology by Dong Min
Kim on Prezi
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Diabetes and Biotechnology
Biotechnology solutions
Recombinant DNA
Questions
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What is diabetes?
Why is there growing concern about
diabetes?
What is the role of insulin?
What is the role of biotechnology in the
treatment of diabetes?
Biotechnology & Diabetes Treatment
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Read Calorie-Coated Diabetes
Beta Cell in Pancreas
Pancreas, thymus
Cell Biology
Of Insulin
Response
Cell Biology
Of Insulin
Response
Cell Biology Of Insulin Response
Insulin
A small, simple protein
Gene is on
the 11th
Chromosome
(short arm)
It contains
153 bases.
Recombinant DNA with insulin gene inserted
Recombinant DNA: A desired gene to
the plasmid. Bacteria is transformed
to accept the plasmid.
Chromosome & Plasmid
Interesting facts about plasmids
What do plasmids do in
bacteria?
 In nature?
Why are plasmids used in
recombinant DNA?
Interesting facts about plasmids
What do plasmids do in
bacteria?
 Extra genes
 Not essential for living
 Benefit for survival under
certain conditions
In nature, some plasmids
 Antibiotic genes
 Antibiotic resistant genes
Why are plasmids used in
recombinant DNA?
 Bacteria can be
transformed by adding
circular DNA but not
linear
 Exonuclease:
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Breaks down genes in a
linear process
Outside to the inside
Making a Recombinant DNA Product
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What are the steps needed?
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Paper plasmids 
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Construction of the pAMP and pKAN Plasmids
Questions
Genetic Engineering –Recombinant DNA
How?
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Identify a molecule produced by a living
organism
Isolate the instructions (DNA sequence =
gene)
Put the instructions into another cell or
organism
Allow the cell to replicate
Harvest the desired product
The Vector (E. coli bacteria).
What is needed to produce a product with
recombinant DNA?
Recombinant DNA
Insulin
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Identify a insulin gene in humans
Use restriction enzymes to isolate the gene in a
DNA fragment
DNA fragment is added to another DNA source =
vector
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such as plasmids of bacteria or yeast
Recombinant DNA is placed in a host cell
As the host cell divides (replicates), the rDNA also
replicates
Harvest, purify, test & market
What we need to learn about:
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Bacteria (Vector)
Copying cells - Mitosis
Proteins
How are enough insulin is made? PCR
Recombinant DNA & Cloning
What is DNA cloning?
• When DNA is
extracted from an
organism, all its genes
are obtained
• In gene (DNA) cloning
a particular gene is
copied (cloned)
Why Clone DNA?
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A particular gene can be isolated and its nucleotide
sequence determined
Control sequences of DNA can be identified &
analyzed
Protein/enzyme/RNA function can be investigated
Mutations can be identified
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e.g. gene defects related to specific diseases
Organisms can be ‘engineered’ for specific purposes
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e.g. insulin production, insect resistance and
more
How is DNA cloned?, I
Blood sample
• DNA is extracted- here
from blood
• Restriction enzymes,
e.g. EcoR I, Hind III,
etc., cut the DNA into
small pieces
• Different DNA pieces
cut with the same
enzyme can join, or
recombine.
DNA
Restriction enzymes
DNA Cloning, II
• Bacterial plasmids (small
circular DNA additional to
a bacteria’s regular DNA)
are cut with the same
restriction enzyme
• A chunk of DNA can thus
be inserted into the
plasmid DNA to form a
“recombinant” molecule
DNA cloning, III
• The recombinant plasmids
are then mixed with bacteria
which have been treated to
make them “competent”, or
capable of taking in the
plasmids
• This insertion is called
transformation
DNA Cloning, IV
• The plasmids have
naturally occurring (or
inserted) genes for
antibiotic resistance
• Bacteria containing
plasmids with these genes
will grow on a medium
containing the antibioticthe others die, so only
transformed bacteria
survive
DNA Cloning, V
• The transformed bacterial
cells form colonies on the
medium
• Each cell in a given colony
has the same plasmid (&
the same DNA)
• Cells in different colonies
have different plasmids (&
different DNA fragments)
Screening, I
Screening can involve:
1. Phenotypic screeningthe protein encoded by
the gene changes the
color of the colony
2. Using antibodies that
recognize the protein
produced by a particular
gene
Screening, II
3. Detecting the DNA sequence of a cloned gene
with a probe (DNA hybridization)
rDNA project
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Choice of format:
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Presentation:
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PowerPoint Presentation with notes
Globster (electronic poster)
Museum boxes
Process to make rDNA
Process before rDNA
Benefits
Disadvantages or bioethical issues
Regulation
Equivalent to Test Grade
Additional Resources
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http://www.accessexcellence.org/AE/AEC/CC/rest
riction.php
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Discovery and Applications of REs http://videos.howstuffworks.com/hsw/22635discoveries-with-bill-nye-restriction-enzymesvideo.htm
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http://tools.neb.com/NEBcutter2/
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http://www.accessexcellence.org/AE/AEC/CC/acti
vity1.php