1. The document describes a lab activity where students will create a model of a recombinant plasmid to demonstrate how they are made and their usefulness. 2. Students will cut out strips representing bacterial DNA and human DNA containing the insulin gene. They will use a restriction enzyme to cut the DNA strands, leaving complementary sticky ends, and fuse the insulin gene into the bacterial DNA to create a recombinant plasmid. 3. Recombinant plasmids allow bacteria to produce useful human proteins like insulin, helping treat diseases like diabetes. Restriction enzymes cut the DNA at specific sites, leaving ends that fuse together to insert human genes into bacterial DNA.

1. 1-15-16 Agenda & Objective
Paper Plasmid Lab
ObjectiveObjective
Create a model of a recombinant plasmid and use it
to explain how they are made and why they are
useful

3. Preparation
Cut out the Cell DNA
(goldenrod). These
must be glued together
in the order indicated at
the bottom.
Keep this DNA in one
long sheet.

4. Preparation
1. Cut out the bacterial DNA
strips – pink paper
2. Toss out any 2 strips, except
for the one with the replication
site
3. Glue your strips together in
any order, end-to-end.
4. Form a ring with them and tape
the ring together.
Does everyoneDoes everyone
have the samehave the same
ring of bacteriaring of bacteria
DNA?DNA?

5. Okay…here is what we are
going to do:
1. We are going to cut out the gene (DNA)
for insulininsulin from some human DNA
2. We are going to cut open the DNA from a
bacteria
3. We are going to fuse the 2 together
4. Then the bacteria will be able to make
human insulin
What gene areWhat gene are
we going to cutwe going to cut
out of theout of the
human DNA?human DNA?
Where areWhere are
we going towe going to
put thisput this
humanhuman
gene?gene?
WHY?WHY?

6. How we will do it
• We need to find the 11
restriction enzymerestriction enzyme that
can cut BOTH DNAs for
us
 Cuts the human DNA 2
times
 Cuts the bacteria DNA 1
time
• Then the DNA pieces will
have the same sticky ends
and will match up to each
other
Human DNA
Bacteria DNA
Bacteria DNA
How manyHow many
enzymes willenzymes will
we end upwe end up
using to cutusing to cut
the DNA?the DNA?

7. How it’s done
• Cut the DNA from both
organisms with the SAME
restriction enzyme
• So they have complimentary
overhanging “sticky ends”
• Combine the DNA with
DNA ligase - an enzyme that
will seal them together

8. Examples
A restriction enzyme may recognize the
sequence
And it cuts after the A on both strands.
What will the cut look like?
ACCGGT
TGGCCA

9. What we use it for
• Put insulin gene into yeast to make insulin
for diabetics
• Factor VIII into bacteria for hemophiliacs
• Human Growth Hormone
• Erythropoietin for treating anemia

10. Procedure
Run each enzyme slowly
up and down each DNA
It must cut your PINKPINK
plasmid only ONCEONCE
If must cut your
GOLDENRODGOLDENROD Human
DNA TWICETWICE – above
and below the gene of
interest.
How manyHow many
enzymes areenzymes are
we going towe going to
end upend up
using?using?

11. Procedure
Select the ONE enzyme that can
cut your bacteria DNA once
and your Cell DNA twice.
Record it’s name in your data
table.
Use scissors to cut the DNA of
the bacteria and the Cell as
this enzyme would, in a
staggered fashion.

12. Procedure
Use tape to “splice” the
goldenrod cell DNAgoldenrod cell DNA
genegene into your pinkpink
bacteria DNA.bacteria DNA.
You have now created
recorecombinmbinantant DDNNAA

14. Analysis Questions
1. In real life, not all bacteria will
successfully take up the cell DNA.
Scientists need a way to select only the
bacteria that have the gene. They do this
by putting antibiotics on the bacteria.
Those that survive must have the
antibiotic resistance gene, along with the
cell DNA.
What antibiotics are your bacteria resistant
to?

15. Procedure
On your bacterial (PLASMID) DNA (the pink
paper):
a. Do you have the gene for ampicillin
resistance?
b. Do you have the gene for kanamycin
resistance?
c. Do you have the gene for tetracyclin
resistance?
d. We ALL must have the replication site

16. Analysis Questions

17. Draw in the location of the
insulin gene