A Simulation in DNA Recombination – Building a Paper Plasmid
1.
Homework: a. Cut out the plasmid strands and tape them all together in
any order. Form a circle.
b. Cut out the Cell DNA strands but place them in order.
Keep this DNA linear.
c. Cut out the restriction enzyme cards.
2.
Class:
Discuss: plasmids, restriction enzymes, recognition sites,
sticky ends, restriction map
a.. Find where recognition sites are present for each RE on the plasmid.
Mark the entire site on the plasmid and identify, by name, the RE that
cuts the site. Keep track using the data sheet.
REMEMBER THAT YOUR GOAL IS TO FIND THOSE ENZYMES THAT
WILL CUT THE PLASMID ONCE AND ONLY ONCE. Continue this procedure
until all 8 enzymes have been tried. Everyone’s results will be different because of
different plasmid sequences. If you have no enzymes that will cut your plasmid only
once, thenreconstruct your plasmid.
b. With the RE that can cut the plasmid, find which of the
same RE cut the Cell DNA.
c. Draw a restriction map (using your data sheet) showing the RE
restriction sites and genes found on the plasmid. A restriction map
may also be drawn for the Cell DNA.
Discuss how RE can be used to insert the DNA of
interest from Cell DNA into the plasmid.
d. Find which RE can be used to cut both Cell DNA and
plasmid so that the Cell DNA's gene of interest can be inserted into
the plasmid. Remember that the protein coding sequence from the
Cell DNA must not be cut nor should the replication origin or one
antibiotic resistant gene be cut on the plasmid. Pick an enzyme
that cuts as close to either end of the gene of interest as possible.
Homework: Finish the engineered plasmid and illustrate a
new restriction map of the newly generated
plasmid below.
Restriction map of new recombinant plasmid:
3. Class: Compare the various constructs made by the base pair size
of the engineered plasmid. Using one students construct, you can
simulate a gel electrophoresis with the engineered plasmid. Cut the
plasmid with the same RE that allowed for recombination to occur and use
the DNA fragments for an explanation of how electrophoresis works. You
can relate the topics of recombinant DNA to cloning and how the
technique is used for massive production of insulin, or other DNA
recombination products.
pGLO Plasmid Map
Our unique pGLO plasmid map is available for educational use only.