MODELING GENE TRANSFER WITH A PLASMID
Introduction
1. Define the following:
Plasmid –
Name ____________________________________
DUE ___________ Per ____ Mrs. Naclerio
Restriction enzyme –
Recombinant DNA –
Sticky ends –
Gene splicing –
The first step in genetic engineering is to incorporate a desired gene into a plasmid. The plasmid and gene must be prepared so they
can be joined together to form a new recombinant plasmid. One way to do this is to cut both the gene and the plasmid with restriction
enzymes that leave overhanging or so called sticky ends. If the sticky ends of the plasmid and the gene are complementary, they will
form hydrogen bonds. The sticky ends can then be joined permanently by ligase enzymes to make a new plasmid containing
recombinant DNA. In this activity you will make a model recombinant DNA plasmid by piecing together a paper gene and a paper
plasmid that have been cut with restriction enzymes.
Objectives
 Explain the function of a restriction enzyme
 Model the process of making a recombinant DAN plasmid from a desired gene and the plasmid
Materials (per group)
 pUC19 Plasmid Sequence
 Lux Gene Sequence
 Scissors
 Colored pencils/crayons
 Tape
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Safety
 Be careful when using the scissors
Procedure
1. Using the pUC19 Plasmid Sequence strip, write out the complementary DNA sequence under the first strand. REMEMBER
THE BASE-PAIRING RULE!!!
pUC19 Plasmid Sequence
…ATGACCATGATTACGCCGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCC…
2. Color this pUC19 Plasmid Sequence blue. Cut out around black line.
3. Tape the two ends together to make a complete circle that represents the pUC19 plasmid, as
shown in Figure 2. Note: Make sure that the DNA sequence of the plasmid is facing outward.
Label this circle plasmid.
4. The actual lux gene makes a chemical that causes an organism to glow in the dark. On the Lux
Gene Sequence (neon orange strip), write the sequence for the complementary DNA strand
directly under the sequence for the first strand, near the bottom edge.
Lux Gene Sequence:
CACGGATCCAGCTTAGAGTACCTAGATAAGGCATGGATCCAGCTACGCAA
5. After you write the lux Gene Sequence, label this strip of paper Lux gene.
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6. Identify the recognition site for the following restriction enzyme on the pUC19 plasmid DNA. The solid lines indicate
where the restriction enzyme has made a cut within the recognition sequence. Draw lines on the blue strip of paper labeled
plasmid to show where the plasmid should be cut by the t restriction enzyme listed below.
7. Identify the recognition sites (2) for the same restriction enzyme above on the lux gene DNA. Draw lines on the neon orange
strip of paper labeled gene to show where the gene should be cut by the restriction enzyme (2) listed below.
8. Use the scissors to cut the blue strip of paper that represents the plasmid and the neon orange strip of paper that represents
the gene according to your cut site markings. Be careful to leave the overhang, or sticky ends, intact.
9. Match the complementary sticky ends from the pUC19 plasmid and the lux gene to form a new plasmid. Tape the blue and
neon orange pieces together to represent the bonds that will form between the complementary strands. The result should be a
circle of neon orange and blue paper. In a real plasmid, the sticky ends are first held together by hydrogen bonds and then
permanent covalent bonds are formed by the ligase enzyme.
10. Tape the neon orange and blue strip of paper and the extra pieces of neon orange to your laboratory record sheet. Then,
answer the questions.
Observations
 Complete the plasmid model
 Be sure to label the plasmid section and the lux gene section of the recombinant DNA
 Be sure to put your name on the model
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Analysis and Conclusions
1. Use the data table below. Relate the steps of producing recombinant DNA to the activities of the modeling procedure by
explaining how the terms relate to the model.
TERM
LAB MODEL
GENE SPLICING
PLASMID
RESTRICTION
ENZYME
STICKY ENDS
RECOMBINANT
DNA
2. How does the paper model of a plasmid resemble a bacterial plasmid?
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3. How is cutting with the scissors different from cleaving with a restriction enzyme? What is specific about this enzyme?
4. Enzymes that modify DNA, such as restriction enzymes, have been discovered and isolated from living cells. What functions do
you think they have in living cells?
5. Does the model accurately represent the process of producing recombinant DNA?
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pUC19 Plasmid Sequence
ATGACCATGATTACGCCGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCC
pUC19 Plasmid Sequence
ATGACCATGATTACGCCGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCC
pUC19 Plasmid Sequence
ATGACCATGATTACGCCGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCC
pUC19 Plasmid Sequence
ATGACCATGATTACGCCGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCC
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Lux Gene Sequence:
CACGGATCCAGCTTAGAGTACCTAGATAAGGCATGGATCCAGCTACGCAA
Lux Gene Sequence:
CACGGATCCAGCTTAGAGTACCTAGATAAGGCATGGATCCAGCTACGCAA
Lux Gene Sequence:
CACGGATCCAGCTTAGAGTACCTAGATAAGGCATGGATCCAGCTACGCAA
Lux Gene Sequence:
CACAAGCTTAGCTTAGAGTACCTAGATAAGGCATGGATCCAGCTACGCAA
Lux Gene Sequence:
CACGGATCCAGCTTAGAGTACCTAGATAAGGCATGGATCCAGCTACGCAA
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