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Name: ___________________________________________ Date: _________________
The E. coli Insulin Factory
Recombinant DNA Activity Packet
Background:
Bacteria have pieces of circular DNA called plasmids along with their normal
DNA. Plasmids are a wonderful ally for biologists who want to utilize bacteria to
produce very specific proteins. The plasmids can be cut, fused with other DNA, and then
reabsorbed by bacteria. The bacteria can easily incorporate the new DNA information
into their metabolism. This “recombining” of DNA is called recombinant DNA.
Extracting a gene from one DNA molecule and inserting it into another requires
precise “cutting and pasting”. To carry out this procedure, a piece of DNA containing the
gene of interest must be cut out of a chromosome and “pasted” into a bacterial plasmid.
The cutting tools for making recombinant DNA are restriction enzymes, which
were first discovered in the late 1960s. They work by chopping up DNA, a process
called restriction because it restricts foreign DNA from surviving in the cell. Your own
DNA has been chemically modified by other enzymes in a way that protects it from the
restriction enzymes.
Most restriction enzymes recognize short nucleotide sequences in DNA molecules
and cut at specific points within these recognition sequences. Several hundred restriction
enzymes and about a hundred different recognition sequences are known.
Goals:
In this activity, a make-believe DNA message for the protein insulin is marked on
the cell DNA. Your task will be to find a restriction enzyme that cuts the plasmid once
(and only once) and the cell DNA as close as possible on both ends of the insulin gene so
that the insulin code can be fused into the circle of the plasmid DNA.
Materials:
Each group will need:
 6 PINK Plasmid DNA strips
 6 GOLDEN Cell DNA strips
 8 GREEN Restriction Enzymes
 1 GREEN Ligase Enzyme
 Scissors
 Tape
Directions:
Part I: Creating Circular Plasmid DNA
Have one partner work on creating plasmid DNA while the other partner moves to part II.
1. Choose 4 of the PINK Plasmid DNA strips to use in creating your Plasmid DNA;
making sure that one of the 4 strips of paper includes the “origin of replication”
section. Place the other 2 PINK Plasmid DNA strips aside.
2. Shuffle the 4 chosen PINK Plasmid DNA strips and tape them end to end in any
random order (as long as the letters are going in the same direction).
3. After you have taped the four strips into one long strip, tape the two remaining ends
together to form one long circular paper plasmid.
4. Set paper plasmid aside until Part III.
Part II: Creating Cell DNA
While one partner is working on Part I, the other partner should create cell DNA.
1. Tape all YELLOW Cell DNA strips together end to end in the order indicated on
each strip of paper.
2. Match the bottom of strip #1 with the top of strip #2, etc.
3. The DNA code for insulin (the protein gene of interest) will be shaded.
4. Once all YELLOW Cell DNA strips have been attached to one another, set aside until
Part III.
Part III: Identify where Restriction Enzymes
will cut the Plasmid DNA.
Overview:
Work together with your partner to determine where the restriction enzymes will cut the
plasmid DNA.
There are 8 GREEN Restriction Enzymes given for cutting the DNA and one
GREEN Ligase used for fusing DNA segments. Note that on each of the restriction
enzyme rectangles, there is the name of the enzyme (such as Ava II) and a short DNA
sequence that shows exactly what sequence that enzyme cuts.
Your job as a biochemist is to find a restriction enzyme that will cut open your
plasmid at one site only. The same restriction enzyme should be able to cut your cell
DNA at two sites, one above and one below the insulin gene. Try to find a restriction
enzyme that will cut as close to the insulin gene as possible, without cutting the gene.
You and your partner have 8 restriction enzymes to choose from. Some of the
enzymes cannot cut open your plasmid, some can. Some of the enzymes cannot cut your
cell DNA at two sites, some can. If a restriction enzyme doesn’t meet ALL of your
needs, then the enzyme is unusable.
Follow the steps listed below and fill in data table / plasmid map (on the answer sheet):
1. One partner should take an enzyme, for example, Ava II, and check the pink
plasmid for a location or locations that can be cut by this enzyme. The other
partner should use a pencil to mark the approximate location on the plasmid map
where each restriction enzyme will cut the plasmid DNA. Also, note on the data
table how many times each restriction enzyme will cut the plasmid. Remember
that your goal is to find the enzymes that will cut the plasmid only ONE TIME.
2. Once you have selected the restriction enzyme or enzymes that will cut the
plasmid one time only, check these restriction enzymes against the cell DNA
strand. Remember, this time the goal is to find which enzyme will cut the cell
DNA as close as possible above and below the insulin gene without cutting into
the gene.
3. Mark directly on the YELLOW cell DNA strip where the enzymes will cut. Draw
the line accurately showing exactly where the bases will be cut. Make sure these
lines are drawn in the staggered manner in which the restriction enzymes will
actually cut, resulting in “sticky ends”. Write the name of the enzymes next to
each line you draw to keep track.
4. After you have completed testing the enzymes and plotting where cuts will be
made, determine which ONE enzyme you would use to cut the plasmid and the
cell DNA. Use scissors to make the cut in your plasmid and the cell DNA. Make
sure to cut the DNA in the staggered way which will expose the “sticky ends”
where joining will be possible. Since only one restriction enzyme was used, all
sticky ends will be complimentary to one another when they are joined together.
5. Use tape to splice your insulin gene into the plasmid chain. You have created
RECOMBINANT DNA!!!!
6. Fill in the rest of the appropriate spaces in the table on the answer sheet. Give
exact reasons why you did not use certain enzymes and the reason why you chose
a particular restriction enzyme.
7. Answer the analysis questions at the bottom of the answer sheet.
Answer Sheet:
Name: __________________________________
Data Table:
Name of
Restriction
Enzyme
Number of cuts on:
Plasmid
Cell
DNA
DNA
Should you use this enzyme in creating the recombinant DNA? Explain.
Ava II
Bam HI
Bgl II
Eco RI
Hin dIII
Hpa II
Sac I
Xma I
Plasmid Map:
Orient your plasmid DNA with
the plasmid map and record / label
all the approximate locations where
each of the 8 restriction enzymes
will cut the plasmid DNA.
Analysis Questions:
1. What did the scissors represent in this activity?
2. Why is it important that the SAME restriction enzyme is used to cut the plasmid
DNA as well as the cell DNA?
3. What enzyme did the tape represent in this activity?
4. What is the resulting molecule of DNA that you created called?
5. What restriction enzyme did you decide to use on your plasmid DNA and cell
DNA? Explain, in detail, why you chose this restriction enzyme over the others
available to you.