Download Recombinant DNA and the Production of Insulin

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Recombinant DNA and the Production of Insulin
Part One
Diabetes is a condition where a person has too much sugar in their blood. Insulin, which is a hormone
created by the pancreas, normally helps lower the level of sugar in a person’s blood. But people who are
diabetics do not produce enough insulin to properly lower their blood sugar. Too much sugar in the blood
can cause negative health effects such as heart disease, high blood pressure, and can lead to a stroke.
Sufferers of diabetes are often required to take insulin injections whenever their blood sugar becomes
elevated. This means they must have access to a steady supply of insulin. It is estimated that over 350
million people around the world suffer from diabetes. Through bioengineering, scientists have isolated the
human gene to produce insulin. Once they found this gene, they removed the DNA sequence.
A. Cut out the six strips of Human DNA and glue/tape them in order to make one long strip. Add strip
2 to the bottom of strip 1. Add strip 3 to the bottom of strip 2. And so on…
B. When finished, you should have one long strip of human DNA.
Part Two
Bacteria have circular pieces of DNA called plasmids. They are a wonderful tool for bioengineers.
Scientists have discovered a way to program bacteria to produce human insulin! The basic idea is fairly
simple. First, the plasmid DNA of the bacteria is removed. Next, using tiny “scissors” the bacteria plasmid
is cut open. Then, the human gene to create insulin is glued into the bacteria plasmid. Finally, the
engineered plasmid is reinserted into the bacteria. The only difference however is the bacterium has now
been programmed to create human insulin. This process of combining DNA of two different organisms
creates “recombinant DNA”. Bioengineers have discovered a way to program bacteria into making human
insulin. When harvested, the insulin can be given to sufferers of diabetes.
C. Cut out the six strips of bacterial plasmids. Glue/tape them in order. Add strip 2 to the bottom of
strip 1. Add strip 3 to the bottom of strip 2. And so on…
D. When finished, glue the strips in the shape of a ring. This represents how the plasmid DNA of
bacteria can be found naturally.
Part Three
RESTRICTION ENZYMES are a special type of protein. Their job is to cut DNA into segments at very
precise locations. There are a number of restriction enzymes that are available to cut the plasmid DNA and
the human DNA. A scientist must identify which restriction enzyme to use based on their goal. In this
activity, our goal is to remove the human gene that produces insulin from the strip of human DNA. In
order to do this, we must cut the human gene just before and just after the start of the gene sequence. That
means that we must find a restriction enzyme to cut the long strip of human DNA twice.
Insulin Gene E. Cut out the three restriction enzymes: Eco R1, Hind III, and Ava II. These three restriction enzymes
are given to cut the Human DNA and Plasmid DNA.
F. Your job as a biochemist is to find a restriction enzyme that will cut your Human DNA at TWO
locations, one above and one below the gene for insulin.
G. Draw and label the location of the cuts on your strip of Human DNA for all three restriction
enzymes.
Did Ava II cut Human DNA just before and just after the insulin gene?
YES or NO
Did Eco R1 cut Human DNA just before and just after the insulin gene?
YES or NO
Did Hin dIII cut Human DNA just before and just after the insulin gene?
YES or NO
Part Four:
Restriction enzymes break apart the bonds that hold the double helix together. In the picture below, you
can see that a restriction enzyme will leave unpaired nitrogen bases behind. These unpaired nitrogen bases
are known as “sticky ends”. They are called “sticky ends” because of Chargaff’s Rules. An unpaired T
will want to stick to an unpaired A. An unpaired C will want to stick to an unpaired G.
Our goal is to find a restriction enzyme that will cut open the circular bacteria plasmid. Because the
plasmid DNA is circular, we need to find a restriction enzyme that will cut open the plasmid at only ONE
location.
Bacteria Plasmid DNA H. Draw and label the locations of the cuts on your circular plasmid of Bacteria DNA for all three
restriction enzymes (Eco R1, Hind III, and Ava II).
Did Ava II cut the Bacteria Plasmid DNA once?
YES or NO
Did Eco R1 cut the Bacteria Plasmid DNA once?
YES or NO
Did Hin dIII cut the Bacteria Plasmid DNA once?
YES or NO
Part Five:
Which restriction enzyme can we use to cut the Human DNA twice and the bacterial plasmid DNA once?
Answer = ______________________________
Remember that when DNA is cut apart by restriction enzymes, sticky ends are left behind. These sticky
ends will be the “glue” that is required to combine the human DNA with the bacteria DNA. Because we
used the same restriction enzyme to cut both the human DNA and the bacterial DNA, the “sticky ends”
should connect and bond together, thus creating RECOMBINANT DNA. This is how bioengineers can
mix the DNA of two different organisms together.
I. Identify the proper restriction enzyme to use.
J. Use scissors to make the cut into the Bacteria Plasmid and the Human DNA. Be certain to make the
cuts in the staggered fashion as this will expose the “sticky ends” where joining will be possible.
K. Use tape/glue to splice you insulin gene into the plasmid chain. You have now created a
RECOMBINANT DNA!!!!
Part Six
In a real situation, you would mix your recombinant DNA with the bacteria of your choice. These bacteria
would hopefully absorb the recombinant DNA. These bacteria should then begin producing insulin. You
could purify the insulin and sell it so that it could be used by diabetics.
How do we know for sure if the bacteria absorbs the recombinant DNA? One way to test is to use
medicines designed to kill bacteria. As you may know, antibiotics are medicines that are designed to kill
bacteria when people have infections. Engineered bacteria will often contain a gene to resist (fight off)
certain antibiotics.
Examine your recombinant DNA. Which antibiotic will your bacteria resist? _______________________
If the bacteria properly absorbs the recombinant DNA, then it should be able to survive in the presence of
penicillin. To check and see if this has happened is simple. You would simply place your engineered
bacteria in an environment with penicillin. If the bacteria survives, it must have absorbed the recombinant
DNA and the process was a success. If the bacteria dies, then something must have gone wrong. If the
bacteria dies, it means the recombinant DNA was never absorbed into the bacteria. The scientist would
have to start over.
Prior to recombinant DNA technology, insulin was obtained from pigs and cows. However, many
diabetics were allergic to this type of insulin. Now that insulin is manufactured from Human DNA, this
problem has been solved.
2 3
T A
T A C G G C T A C G A T T A G C T A G C C G C G T A T A T A T A A T A T A T T A 4
Gene 1 T A
T A C G G C A T A T G C G C T A A T C G A T T A A T A T C G G C T A C G T A C G G C A T G C A T T A T A C G T A T A A T A T G C T A C G A T A T G C C G A T G C G C T A G C G C G C C G C G T A A T G C G C C G A T C G A T G C G C G C C G C G C G G C Insulin Human DNA G C T A A T A T T A A T T A T A C G C G T A C G C G T A T A A T A T G C A T A T T A T A
T A C G G C A T A T C G G C G C G C G C C G C G C G T A A T G C G C A T C G C G 5 6
1 A T G C A T A T A T A T T A G C T A G C T A G C T A C G C G A T G C T A A T G C G C Penicillin (antibiotic) G C C G C G C G A T G C A T G C T A T A T A C G T A T A A T A T G C G C T A C G T A 2 Resistance gene Bacterial Plasmid DNA T A
A T G C G C C G C G C G C G C G T A T A T A T A T A A T G C G C G C A T C G T A C G
G C A T G C T A T A A T A T C G C G T A A T G C G C A T G C G C G C C G C G C G T A G C G C T A G C G C G C G C G C C G A T A T G C G C T A T A A T T A A T C G T A T A
A T A T G C C G C G G C T A A T G C G C T A T A C G G C A T A T C G T A C G C G 3
4
5 6
Restriction Enzymes C G C G T A Ava II G C G C T A C G T A T A C G Hin dIII T A Eco R1 G C A T A T A T A T G C Restriction Enzymes C G C G T A Ava II G C G C T A C G T A T A C G Hin dIII T A Eco R1 G C A T A T A T A T G C