Download RECOMBINANT DNA USING BACTERIAL PLASMIDS

Name:_________________________
RECOMBINANT DNA USING BACTERIAL PLASMIDS
BACKGROUND:
Bacteria have not only their normal DNA, they also have a circular DNA
called a plasmid. It is a wonderful ally for biologists who desire to get
bacteria to produce very specific proteins. The plasmids conveniently can
be cut, fused with other DNA and then reabsorbed by the bacteria. The
bacteria easily incorporate the new DNA information into its metabolism. This “recombining” of DNA
is called RECOMBINANT DNA.
GOALS:
In this activity, a make-believe DNA message for the insulin gene is marked on the human DNA. Your
task will be to find an enzyme that cuts the bacterial plasmid once (and only once) and the human
DNA as close as possible on both ends of the insulin gene so that the insulin gene can be
fused into the circle of plasmid DNA. This way the bacteria will start producing human
insulin.
INSTRUCTIONS:
1. Pick a partner and collect all necessary papers. Obtain scissors and a strip of tape about 8
inches in length.
2. Cut out the PLASMID (pink) strips. Discard any two of the strips
(except for the strip which contains the replication origin site).
Shuffle the remaining strips and tape them end-to-end in any
random fashion. Tape the two remaining ends together to form a
ring of paper.
3. Cut out the human DNA (blue) strips. They must be taped
together in the order indicated at the bottom (i.e. strip 2 is taped
to the bottom of strip 1, strip 3 is taped to the bottom of strip 2,
strip 4 is taped to the bottom of strip 3, etc).
4. After completing steps 2 and 3, use the PLASMID MAP SHEET
(yellow) to mark the relative locations of the genes for antibiotic
resistance in your plasmid using a pencil.
5. You are now ready to pick an enzyme to cut the DNA. Your job as a scientist is to find a
restriction enzyme that will cut open your plasmid at ONE site only (this may or may not be
possible depending upon how you constructed your plasmid). The SAME enzyme should be
able to cut your human DNA at TWO sites, one before the insulin gene and one after the
insulin gene. Also, you must try to cut the human DNA as close to the insulin gene as possible.
a. Cut out the ENZYMES (green).
Name:_________________________
6.
7.
8.
9.
b. You have 8 enzymes to choose from. Some of the enzymes cannot cut open your
plasmid, some can. Some of the enzymes cannot cut your human DNA at two sites.
You will NOT be able to use those enzymes.
c. Start with the first enzyme, AVA II, and check the pink plasmid for a location(s) which
can be cut by this enzyme. Use a pen to mark on the pink plasmid where the enzymes
will cut. Write the name of the enzyme that would make the cut next to the line you
have drawn on the plasmid. Also, use the data table on the yellow answer sheet to
record how many times AVA II will cut the plasmid. REMEMBER, YOU WANT AN
ENZYME THAT WILL ONLY MAKE ONE CUT INYOUR PLASMID (IF POSSIBLE). Continue
doing this with all 8 enzymes.
d. Now check the 8 enzymes for possible cut sites on the human DNA. REMEMBER, THE
GOAL IS TO FIND AN ENZYME THAT WILL MAKE A CUT CLOSE TO THE GENE FOR
INSULIN, ONE BEFORE THE GENE AND ONE AFTER. Use a pen to mark on the blue
human DNA where the enzymes will cut; write the name of the enzymes next to each
line you draw.
After you have tested all 8 enzymes, decide which ONE enzyme you would choose to cut the
plasmid and the human DNA. Use the scissors to make the cut in your
plasmid and cell DNA in the staggered fashion made by the actual
enzymes. These are called STICKY ENDS. Use tape to splice you insulin
gene into the plasmid. Ligase is the enzyme used to fuse the sticky
ends together, it is like the tape that you are using. YOU HAVE
CREATED A RECOMBINANT PLASMID.
Fill in the appropriate spaces on the yellow sheet, giving reasons why
you did not use certain enzymes and the reason why you chose the
enzyme you did decide upon. (note, the yellow sheet has questions on
the front and the back!)
In true life, you would mix your recombinant plasmids with host bacteria. The host bacteria
would take in your recombinant plasmids, multiply and grow, and begin producing human
insulin. You could purify the insulin and sell it so that it could be used by diabetics.
What is the purpose of using plasmids with genes for antibiotic resistance?
a. The host bacteria are normally killed by antibiotics (Kanamycin, ampicillin, and
tetracycline). However, IF the recombinant plasmids (which contain genes for
antibiotic resistance) have actually been taken up by the host bacteria, then the
bacteria could survive if placed in a growth medium containing an antibiotic. Any
hosts that failed to take the recombinant plasmids would die in such a growth
medium. THEREFORE…..the host bacteria could be grown in a culture medium
containing antibiotics…..the bacteria that survive must have taken in your
recombinant plasmids. HOPEFULLY, the gene for human insulin will be
included in the recombinant plasmid, turn on, and produce human
insulin!!
Name:_________________________
Post lab questions:
1.
What is a plasmid?
2. What are restriction enzymes used for in nature?
3. What is meant by a “sticky end?” What is the name of the enzyme used in nature to fuse DNA
together?
4.
How is “exogenous” (DNA from a different organism) used to create recombinant DNA in an
organism?
5.
What at least two ways that recombinant DNA is a useful tool for biotechnology (and
therefore improves society)?
Name:_________________________
1. Which of the antibiotic resistances does your plasmid contain?
2. Which antibiotic(s) could you use in your growth medium to test for plasmid uptake?
3. Which antibiotic(s) could NOT be used in your growth medium to test for plasmid uptake?
Explain why.