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Honors Biology
Name:____________________
Date:________ Period:______
Transformation
Introduction: Transformation is the process by which a bacterium’s DNA is altered to include foreign genes from a
different species. Such transgenic bacteria are used in medical manufacturing facilities around the world. Local facilities
such as Life Technologies in Carlsbad have huge transgenic bacterial farms that produce various human hormones and
enzymes used to treat various ailments such as diabetes. Before the development of transgenic bacteria, patients that
needed insulin were given horse, cow, or pig insulin. The foreign protein was not as efficient and was incredibly
expensive to obtain. Now human insulin is produced in massive volumes at low cost by genetically engineered bacteria.
In this lab you will simulate the techniques used by genetic engineers to create recombinant DNA that codes for the
production of human insulin.
Materials: Graph paper, color pencils (red, blue, green and yellow), scissors, transport tape.
Procedure:
1. Copy the DNA sequences onto graph paper, putting one letter from the DNA sequence into each square:
A-A-G-C-T-T-A-T-G-G-T-C-C-C-G-G-A-C-G-A-A-G-C-T-T-C
2. In the squares below the single strand of DNA, write in the complementary DNA sequence.
3. Color the square for each base utilizing the following color code:
Adenine (A) = red;
Thymine (T) = blue;
Cytosine (C) = yellow;
Guanine (G) = green
4. What parts of the DNA molecules are not represented on this graph paper model? ________________________
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5. Using scissors cut out the double stranded DNA model. Save the unused portion of your graph paper for step 8).
6. The model you have just created represents the insulin gene. Notice that your model contains 20 DNA base
pairs. The actual gene contains 1430 DNA base pairs.
7. Now that the insulin gene has been identified and isolated, what do you think is the next step in creating
recombinant DNA?____________________________________________________________________________
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8. Using the unused portion of your graph paper, copy the following DNA sequence:
G-G-A-T-C-C-T-G-A-C-A-C-C-G-G-C-G-C-G-T-C-A-A-G-C-T-T-C-C-C
9. In the squares below the single strand of DNA, write in the complementary DNA sequence.
10. Color the squares for each base using the previous color code.
11. Cut out your double stranded DNA. Label the back then tape both ends together to create a circle.
12. You now have a plasmid. Recall that plasmids are small rings of DNA found in bacteria.
13. To insert the insulin gene into the plasmid, you must create what genetic engineers call “Sticky ends.” Sticky
ends are unpaired bases at ends of DNA molecules that have been cut apart. Genetic engineers use special
enzymes called restriction enzymes to cut apart DNA molecules. There are a variety of restriction enzymes such
as EcoRI and HindIII. You will use HindIII, which cuts DNA strands between adjacent A’s in the base sequence
shown below:
14. On your plasmid, find the base sequence shown above. Using your scissors separate the double-stranded DNA
ring just as HindIII would. Which bases make up your sticky ends? ______________________________________
15. Find the base sequence above on your insulin gene. You should find two such sequences – one at each end of
the molecules. Cut apart the two DNA strands just as HindIII would. Set aside the leftover bases. What bases
make up the sticky ends on your insulin gene after treatment with HindIII? ______________________________
16. Compare the sticky ends of the plasmid with those of the insulin molecule. What observations can you make?
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17. Move the insulin gene to the open ends of the plasmid. Fit the insulin gene into the ring so the complimentary
bases line up. When you are sure the base pairing is correct, tape the ends to create one new, closed ring. The
enzymes that connect the pieces of DNA are called ligases. Scissors are used to simulate restriction enzymes.
What does the tape simulate? ___________________________________________________________________
18. What is the new plasmid (plasmid + insulin) called? _________________________________________________
19. The next step in genetic engineering would be to insert the altered plasmid into a bacterium.
20. What happens when the bacterium containing the recombinant DNA reproduces? ________________________
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21. Once inside the bacterium, the altered plasmid directs production of human insulin. Because bacterium has no
need for the hormone, it is excreted from the cell. The bacteria reproduce freely in test tubes and/or tanks and
scientists harvest the hormone and use it to treat children patients with diabetes.
Conclusion:
1. How did you know that your insertion of the insulin gene into the plasmid was correct?
2. The restriction enzyme HpaII cuts DNA between adjacent C’s in the following base sequence:
What sticky ends would result if you used this enzyme to cut DNA?
3. Could you have used HpaII to create a recombinant DNA molecule? Explain.
4. The restriction enzyme BamHI cuts DNA between adjacent G’s in the following base sequence:
What sticky ends would result if you used this enzyme to cut DNA?
5. Could you have used BamHI to create a recombinant DNA molecule? Explain.