Activity 4.1.2 Protein Factories
Introduction
For years, Diana Jones relied on insulin shots to get her through the day. Countless needle
sticks measured her sugar levels and alerted her to the need for more medication. Even though
life is a bit simpler now that she has her insulin pump, Mrs. Jones’s survival is still directly linked
to the medicine being pumped into her bloodstream. What she does not realize, however, is that
the protein that is keeping her body regulated can be produced by tiny living organisms,
particularly bacteria.
When most people hear the word bacteria, they probably think about getting sick or about
destroying these invaders with antibiotics. However, bacteria are a very helpful tool in genetic
engineering and in the production of medication. These living cells replicate quickly and their
unique structure provides scientists with the ability to “highjack” their molecular machinery and
insert a new gene – the code for a new protein. Genetically engineered bacteria can be used as
tiny living pharmaceutical factories and can produce many medications and drugs that help
maintain health and homeostasis in the human body.
Remember that a gene is a piece of DNA that provides the instructions for making a protein.
Genes can be cut out of human, animal or plant DNA and placed into bacteria in small rings of
DNA called plasmids. Bacteria in nature can pass plasmids back and forth to share beneficial
genes. In Lesson 1.2 you learned that this swap of genetic material can lead to shared traits
such as antibiotic resistance. In the lab, scientists can alter plasmids to include the code for a
protein of interest and insert this ring of DNA into bacterial cells. The result of this genetic
engineering is that the recipient organism now has the instructions to make a new protein. In
1978, scientists from the California based company Genentech succeeded in manipulating
bacterial DNA to produce human insulin. The final product was released to the public in the
early 1980s, bringing relief to many diabetics.
As you recall from Unit 1, scientists can use the tools of molecular biology to “cut and paste”
genes of interest into bacterial plasmids. These plasmids can then be inserted into bacteria
through the process of bacterial transformation. Once the new gene is inside the cell, the
bacteria multiplies and the new bacterial clones crank out many copies of a desired protein
product, such as insulin. In this experiment, you will not be using bacterial cells to produce
insulin. Instead, you will use bacterial transformation to insert the genetic information necessary
to produce another protein, green fluorescent protein (GFP), into the cells. Since this newly
produced protein glows a bright green, it is easy to gauge the success of the experiment and
really see the process of transformation at work.
Equipment
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Bio-Rad pGLO Bacterial Transformation Kit
o E. coli bacteria
o pGLO™ plasmid
o LB bacterial growth plates
o LB bacterial growth plates with
ampicillin
o LB bacterial growth plates with
ampicillin and arabinose
o Disposable sterile pipets
o Microcentrifuge tubes
o Foam racks
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Medical Interventions Activity 4.1.2 Protein Factories – Page 1
o CaCl2 transformation solution
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Computer with Internet access
Laboratory journal
Transformation Kit – Quick Guide
handout
Transformation Efficiency worksheet
Handheld UV light or transilluminator
Microcentrifuge tube rack
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o Inoculation loops
Water bath – 42°C
Incubator – 37°C
Permanent black marker
Ice and ice container
Colored pencils or markers
Sterile water (optional)
Procedure
Part I: Bacterial Transformation
1. Turn to the diagram of a bacterial cell you drew and labeled in your laboratory journal in Unit
1. Review the function of key structures in this cell and locate a plasmid.
2. Define the term plasmid in your laboratory journal. Write a list of at least three reasons why
plasmids and bacterial cells are a helpful tool in genetic engineering. Use the Internet or your
notes from Unit 1 if you need additional information.
3. View the animations located at the Explore More – Iowa Public Television site
http://www.iptv.org/exploremore/ge/what/insulin.cfm to learn how recombinant DNA
techniques and plasmids can be used to produce human insulin. Outline the steps of the
process in your laboratory journal.
4. Answer Conclusion questions 1-3.
5. Brainstorm how you could use recombinant DNA technology to make normal E. coli bacterial
cells glow green. Where in nature could you find a source for a glowing protein? Discuss
your ideas with a partner.
6. Note that in this laboratory experiment, the plasmid containing the gene of interest, GFP, has
already been produced for you using recombinant DNA techniques. You will complete
bacterial transformation to move this plasmid into bacterial cells. With the new gene,
bacterial cells can now produce the green fluorescent protein.
7. Visit the Dolan DNA Learning Center DNA Interactive site found at
http://www.dnai.org/b/index.html to review the process of transformation.
8. Click on the Techniques tab at the bottom of the screen and then choose Transferring and
Sorting.
9. View the Transformation animation. Take notes in your laboratory journal as you view the
animation.
10. Summarize the goal of the experiment in a well-crafted paragraph in your laboratory journal.
Your summary should include the terms listed below. Underline each term.
o
o
o
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o
o
Aequorea Victoria
jellyfish
gene
pGLO
plasmid
green fluorescent protein
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ampicillin
arabinose
E. coli bacteria
ultraviolet light
chemical transformation
heat shock
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Medical Interventions Activity 4.1.2 Protein Factories – Page 2
11. Draw the pGLO plasmid map, the diagram showing the location of important genes, in your
laboratory journal. Label each of the three key genes in a different color. Provide a one
sentence description of the function of each gene. Make sure to mention how the gene
relates to the protein that is being produced.
12. Answer Conclusion questions 4-5.
13. Obtain a Transformation Kit – Quick Guide handout and the following plates from your
teacher:
o (1) LB bacterial growth plate
o (2) LB bacterial growth plates with ampicillin
o (1) LB bacterial growth plate with ampicillin and arabinose
14. Follow the directions on the Quick Guide reference sheet to complete the experiment. Use
the drawings located on the right side of the page as a visual reference for each step. Make
sure to clearly label each plate. Note whether the media contains ampicillin or arabinose (or
both) and whether you are plating out bacteria that contain the pGLO plasmid or that do not
contain the plasmid. Depending on the length of your lab period, your teacher may ask you
to stop at Step 9. The sample tubes can be incubated overnight at room temperature and the
experiment resumed on the following day.
15. At the conclusion of the experiment, stack your labeled plates and place the stack inverted in
the 37°C incubator. Allow bacteria to incubate for approximately 24 hours.
16. Predict which of the four plates should show growth. Predict which of the four plates should
glow green when exposed to UV light. Write your predictions down in your laboratory journal
and explain your reasoning.
17. Remove the stack of plates from the incubator after 24 hours.
18. Copy the data table shown below into your laboratory journal and follow Steps 21 - 23 to
make observations and to fill in laboratory data.
Growth? (Y/N) If yes,
include a colony count.
Glowing? (Y/N)
Other Observations
-pGLO LB
-pGLO LB/amp
+pGLO LB/amp
+pGLO LB/amp/ara
19. Observe the bacterial growth on each plate. Carefully count the number of colonies that are
visible. You may want to use a black marker to dot each colony you count. This technique
may help you track the colonies you have already counted.
20. Place each plate on the UV transilluminator or use a handheld UV light to determine if the
colonies do or do not glow green.
21. Record any other relevant observations in your data table.
22. Compare the results to the predictions you documented in your laboratory journal. Discuss
your results with the class.
23. Answer Conclusion questions 6-9.
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Medical Interventions Activity 4.1.2 Protein Factories – Page 3
Part II: Calculating Transformation Efficiency
The first genetically engineered human insulin was produced in the early 1980s. This technology
was a huge achievement in diabetes therapy as it allowed for the production of nearly unlimited
quantities of human insulin. In order for the process to work efficiently, it is necessary to
transform as many cells as possible in each round of bacterial transformation. The more cells
that are successfully transformed to produce the desired protein, the more patients can be
helped with the therapy.
Transformation efficiency is a quantitative measurement that gives scientists an indication of
how successful they were in getting the desired DNA into the bacterial cells. Transformation
efficiency in this experiment represents the total number of bacterial cells that express the new
protein (in your case, GFP), divided by the total amount of DNA used in the experiment. This
value tells scientists the number of bacterial cells transformed by one microgram of DNA and is
calculated by the general formula:
Transformation efficiency = Total number of cells growing on the plate
Amount of DNA spread on the plate (in µg)
Before you can calculate the efficiency of your experiment, you will need two pieces of
information: the total number of glowing colonies on your LB/amp/ara plate and the total amount
of pGLO plasmid DNA spread on the LB/amp/ara plate. You counted the number of colonies in
part I of the experiment. Follow the directions below to determine the amount of DNA spread
and to calculate the overall transformation efficiency.
24. Obtain a Transformation Efficiency worksheet from your teacher.
25. Follow the steps on the worksheet to complete the calculations. Use procedural information
found in the Quick Guide as well as the data table from Part I of the laboratory experiment to
assist you with your calculations.
26. Write the formula for transformation efficiency in your laboratory journal.
27. Clearly show your final calculations for transformation efficiency in your laboratory journal.
Report your final value using scientific notation.
28. Note that transformation efficiency values for the experiment completed in Part I are
generally between 8.0 x 102 and 7.0 x 103 transformants per microgram of DNA. Compare
the transformation efficiency of your experiment to this value and describe your findings in
your lab journal.
29. Compare your transformation efficiency to that of another lab group.
30. Answer Conclusion questions 10 and 11.
Part III: GFP as a Medical Intervention
31. View the Virginia Commonwealth University Secrets of the Sequence video A Green Light
for Biology – Making the Invisible Visible at
http://www.pubinfo.vcu.edu/secretsofthesequence/playlist_frame.asp to explore how
the protein GFP can be used to “tag” proteins of interest.
32. Read the article Lighting Up the Lab found at the UCSC Science Communication program
site at http://sciencenotes.ucsc.edu/0001/glow.htm.
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Medical Interventions Activity 4.1.2 Protein Factories – Page 4
33. Use the information from the video and the article to list and describe at least three useful
applications of GFP in cellular biology and medical science in your laboratory journal. Think
about how GFP qualifies as a medical intervention.
34. Answer the remaining Conclusion question.
Conclusion
1. Why are bacteria a good choice for producing many copies of a particular gene in a short
period of time?
2. How are restriction enzymes and ligase utilized in recombinant DNA technology?
3. Explain why it is necessary to use the same restriction enzyme to cut the desired gene from
the source and to cut the plasmid. Include a drawing or diagram in your answer.
4. Explain the role of arabinose in the bacterial transformation experiment.
5. How do the heat shock and the calcium chloride assist plasmid insertion in this chemical
transformation? Relate your answer to the charge on DNA and to the charge on the plasma
membrane of a cell.
6. How do the results of this experiment illustrate the relationship between DNA, proteins and a
trait?
7. How would a genetic engineer distinguish between the bacteria containing the new plasmid
DNA and those bacteria lacking this plasmid DNA?
8. Very often an organism’s traits are influenced by a combination of its genes and its
environment. What two factors must be present in the bacteria’s environment for you to see
the color green? Explain your answer.
9. What advantage would there be for an organism to be able to turn on or off particular genes
in response to certain situations?
10. Provide at least two explanations for why transformation efficiency varied from group to
group. Think about sources of error in the experiment. What could you do to maximize
transformation efficiency?
11. Explain how a value such as transformation efficiency might relate to the business side of
using bacteria to produce proteins.
12. The bacterial cells produce many more proteins than just the GFP. How do you think we can
separate the GFP from the other proteins in the cell?
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Medical Interventions Activity 4.1.2 Protein Factories – Page 5