NAME __________________________
LAB
Recombinant DNA Simulation
(Modified from Boulay/Joslin)
INTRODUCTION
Genetic engineering is possible because of special enzymes that cut DNA. These enzymes are
called restriction enzymes. Restriction enzymes are proteins produced by bacteria used to
prevent (or restrict) the invasion of viruses. They act as “DNA scissors”, cutting viral DNA into
pieces so that it cannot function.
Restriction enzymes recognize and cut at specific places along a DNA molecule called restriction
sites. Each restriction enzyme has its own restriction site. In general, a restriction site is a
4–8 base pair sequence of DNA. The DNA sequence below is the restriction site for the
restriction enzyme known as EcoR1. Its name comes from the bacterium in which it was
discovered: Escherichia coli (E. coli).
5’ – GAATTC – 3’
3’ – CTTAAG – 5’
The EcoR1 enzyme scans a DNA molecule, and each time it finds the sequence above, it makes
one cut between the G and A in each of the DNA strands (see below). After the cuts are made,
the DNA is held together only by weak hydrogen bonds between the four bases AATT. These
bonds are easily broken apart.
Cut Sites
5’ – GAATTC – 3’
3’ – CTTAAG – 5’
Notice the EcoR1 cut sites are not directly across from each other on the DNA molecule. When
EcoR1 makes its cuts, it leaves single-stranded “tails” on the new ends (see above). This type of
end is called a STICKY END because complementary DNA sticky ends can be easily rejoined.
In this activity, we will simulate the production of INSULIN – a hormone made by cells of the
pancreas that regulates carbohydrate metabolism. Individuals with diabetes mellitus require
supplements of insulin to make up for their body's inability to produce sufficient amounts.
To achieve our goal, we need to collect plasmids from E. coli cells. Plasmids are small circular
pieces of DNA found in prokaryotic (bacterial) cells. Next, we need to extract the normal
insulin gene from a human chromosome (chromosome 11). Insulin is made up of TWO
polypeptide chains (51 amino acids). 153 DNA nitrogen bases constitute its gene sequence.
MATERIALS


Paper strip of bacterial plasmid DNA & paper strip of human insulin gene (small portion)
Scissors, tape, highlighter, pencil
METHODS
PLASMID DNA (Bacteria)
1. Cut out the paper plasmid DNA along the rectangular border.
2. Fold each end vertically along the dotted line.
3. Tape the ends together so the plasmid is circular and the DNA bases face outward.
INSULIN GENE EXTRACTION (Human)
4. Cut out the paper human insulin gene along the rectangular border.
5. Locate the TWO EcoR1 restriction sites on the gene and highlight the bases. Mark the cuts
that will be made with pencil. The restriction site is:
5’ – GAATTC – 3’
3’ – CTTAAG – 5’
6. Cut the human DNA with scissors at the 2 restriction sites (see the Introduction for proper
cut sites). The human insulin gene is the DNA found BETWEEN the 2 restriction sites.
INSULIN GENE INSERTION into the PLASMID
7. Scan the plasmid for the single EcoR1 restriction site. Highlight the bases.
8. Cut the plasmid open at this site. If you make an error, tape up your plasmid and try again.
9. Using the rules for base pairing, insert the human insulin gene into the plasmid. Use tape to
seal the ends together.
RESULTS

Your finished product is known as
recombinant DNA since it contains DNA
from more than one organism.

If this were a real experiment, the
recombinant plasmid would then be inserted
into bacteria and the bacteria would be
allowed to reproduce asexually (cloning).

Show your teacher your recombinant plasmid
and have him/her initial your lab handout.
DISCUSSION
1. What is genetic engineering?
(pg. 226)
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2. What is recombinant DNA (hybrid DNA)?
(pg. 226)
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3. What chemical did the “scissors” represent in this experiment?
----------------------------------------------------------------------------------------------------------------------4. DNA ligase is an enzyme used to fuse (or anneal) two DNA fragments together.
What material represented DNA ligase in this experiment?
----------------------------------------------------------------------------------------------------------------------5. How do bacteria use restriction enzymes to protect themselves against viral infections?
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------6. Why was the human insulin gene able to be so easily inserted into the cut bacterial plasmid?
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------7. Imagine your biology teacher gave you another bacterial plasmid with TWO restriction sites
that EcoR1 would recognize/digest (instead of just the one seen in this lab).
How many DNA fragments would you get? ________
8. If scientists wanted to produce human insulin, where should the recombinant plasmid you
created go next?
----------------------------------------------------------------------------------------------------------------------9. What process must occur in the bacteria in order to make thousands of copies of the
recombinant plasmid?
----------------------------------------------------------------------------------------------------------------------10. What will the bacteria be able to produce once this recombinant plasmid is inserted?
-----------------------------------------------------------------------------------------------------------------------
Human Insulin (2 polypeptide chains)