Background
Bacterial have circular plasmids that are usually several thousand base pairs in length.
Plasmids are used in recombinant DNA technology to transfer genes from one organism to
another. A plasmid will have an origin of replication site and may also contain genes for
antibiotic resistance.
Recombinant DNA contains DNA from more than one organism. For example, the gene for
human insulin can be inserted into bacteria with the help of a plasmid. The bacteria will then
secrete human insulin that can be harvested economically and with great purity. The millions of
diabetics throughout the world can then use the insulin.
You have been provided with a paper plasmid and a piece of cellular DNA. The DNA contains a
gene called Green Fluorescent Protein (GFP) and a gene for resistance to the antibiotic
ampicillin. If these genes are inserted into the plasmid, then the bacteria that have the plasmid
will:
1. Be resistant to the antibiotic ampicillin.
2. Be able to glow IF the bacteria have been fed arabinose sugar. Arabinose sugar
activates the GFP gene, which causes the bacteria to glow.
How are these genes inserted into the bacterial plasmid to begin with? These genes were cut
from DNA and inserted into the plasmid. Your task today is to model this process by:
1. Finding the best restriction enzyme to cut out the GFP and antibiotic resistance (shaded
on the cellular DNA).
2. Cutting the plasmid so the gene of interest can be inserted into it.
Part A: Modeling Recombinant DNA Technology
1. Study your plasmid DNA. Notice there are sequences that give antibiotic resistance to bacteria from
antibiotics. There is also an origin of replication site. If any of these sequences are cut, the ability of
the bacteria to reproduce or to have antibiotic resistance will be destroyed.
2. Construct your plasmid by cutting the strips of the
plasmid DNA along the lines. Tape the ends together
to form a circular plasmid. Tape them together in the
same order they are on the paper. For example, the
bottom of strip number 1 would be taped to the top of
strip number 2.
3. Construct your strip of cellular DNA by cutting out the
strips and taping them together in a linear piece. Again,
be sure to tape them in the order they are on the sheet.
Sequence
Shading on Plasmid
Ampicillin
Kanomycin
Tetracycline
Origin of Replication
4. Run each restriction enzyme card along the plasmid.
Origin of Replication
Mark any matching restriction sites you find using a
different color for each enzyme. The restriction enzymes may cut once, more than once, or not at all.
Complete the table in analysis question #1 as you work.
5. Run each restriction enzyme card along the cellular DNA. Mark any matching restriction sites you find
using a different color for each enzyme. The restriction enzymes may cut once, more than once, or
not at all. Complete the table in analysis question #1 as you work.
6. Now you must decide which restriction enzyme to use in your pretend transformation. Here are the
requirements:



Use a restriction enzyme that can cut out the gene of interest (shaded on the cellular DNA).
You want to cut as close to the gene of interest on the cellular DNA as you can so that little
extraneous DNA will be included.
You must also choose a restriction enzyme that can cut the plasmid only once so the
gene can be inserted into it.


Chose a restriction enzyme that does not cut in a gene for antibiotic resistance or in the origin of
replication.
You must use the same restriction enzyme to cut the plasmid as you use to cut out the
gene of interest. This is necessary so the sticky ends will pair up correctly.
7. Now take a scissors and cut the plasmid at the restriction enzyme site selected. Then, cut out the
gene of interest from the cellular DNA at the sites selected.
8. Insert the gene of interest into the plasmid and use tape it into place. This tape represents the
enzyme ligase which is used to “tape” DNA strands together. You should now have a recombinant
pGLO plasmid that has new DNA inserted into it.
Part B: Observations, Analysis, and Conclusions: Answer all questions in complete
sentences.
1. Copy the data table below onto your paper. Fill in this table after completing steps 4 and
5 in Part A.
Restriction Enzyme
Number of Cuts
Made on Plasmid
Number of Cuts
Made on Cellular DNA
Ava II
HindIII
Bam HI
Bgl II
Hpa II
EcoR I
Sac I
XmaI
2. Which restriction enzyme did you choose to use? Why?
3. Specifically explain why EACH of the other restrictions enzymes was not selected for
use.
CELL DNA
T
G
G
G
C
C
T
A
G
G
C
A
C
A
G
G
G
C
C
C
G
A
C
C
C
G
G
A
T
C
C
G
T
G
T
C
C
C
G
G
G
C
1
G
A
G
C T
T T
C C
T G
A A
A A
G G
A G
A T
T A
T C
C A
A T
G A
T A
T C
C G
G T
T C
C T
C C
A
T
T
C
T
T
A
A
G
T
C
A
A
G
C
A
G
G
2
A
A
G
C
T
T
C
C
A
T
G
T
A
T
T
G
C
A
G
A
G
3
T
T
C
G
T
C
A
T
G
T
G
C
C
T
T
T
T
A
A
A
T
A
A
G
C
A
G
T
A
C
A
C
G
G
A
A
A
A
T
T
T
A
4
G
T
A
A
T
A
T
T
C
C
T
C
C
T
T
A
A
G
A
A
T
C
A
T
T
A
T
A
A
G
G
A
G
G
A
A
T
T
C
T
T
A
5
T
T
C
G
A
A
C
G
G
G
G
C
C
C
T
A
G
G
A
C
C
A
A
G
C
T
T
G
C
C
C
C
G
G
G
A
T
C
C
T
G
G
6
PLASMID
G
C
C
C
A
G
A
G
T
T
T
C
T
T
A
A
G
G
T
C
T
C
G
G
G
T
C
T
C
A
A
A
G
A
A
T
T
C
C
A
G
A
1
A
G
A
A
A
A
T
G
T
G
T
G
T
C
C
A
G
T
A
G
G
T T
C A
T G
T G
T C
T C
A C
C C
A C
C T
A T
C T
A T
G T
G A
T G
C G
A G
T A
C C
C T
2
A
T
C
C
G
G
G
G
G
A
A
A
A
A
T
C
C
C
T
G
A
3
C
G
A
G
T
T
A
A
C
C
T
A
G
G
A
G
G
G
C
C
C
G
C
T
C
A
A
T
T
G
G
A
T
C
C
T
C
C
C
G
G
G
4
T
G
G
T
G
G
G
G
G
C
A
A
G
G
T
T
A
T
A
C
T
A
C
C
A
C
C
C
C
C
G
T
T
C
C
A
A
T
A
T
G
A
5
T
A
A
G
C
C
G
T
A
G
G
T
T
C
G
A
A
C
T
C
C
A
T
T
C
G
G
C
A
T
C
C
A
A
G
C
T
T
G
A
G
G
6
ENZYMES (add cut sites)
C
G
T
T
C
G
C
G
T
G
A
C
Ava II G
A
C
T
A
G
T
C
G
C
A
T
G
C
T
C
T
A
G
A
A
G
A
T
C
T
G
G
C
C
C
C Hpa II
G
G
C
T
T
A
A
G
G
A
A Eco RI
T
T
C
C
T
C
G
A
G
G
A
G
C
T
C
G
G
G
C
C
C
C
C
C
G
G
G
Bgl II
Sac I
A
A
Hind
III
Xma I
C
C
G
G
T
A
Bam
HI