A Teaching Module Integrating Literature and Genetic Engineering

Mari Knutson Lynden Public High School

1201 Bradley Rd. Lynden, WA 98264

Summer, 2004

WSU Mentor: Ryan Soderquist, Dr. James Lee Department of Chemical Engineering

Washington State University Pullman, WA 99164-2710

National Science Foundation Grant No. EEC-0338868 supports this project.

Introduction

Overview. This module is designed to encourage students to read and to introduce them to the biotechnology and principles of genetic engineering.

A fiction novel (Robin Cook’s “Chromosome Six”), a fictitious short story (“Bill Schwan’s “Ethics of the Prophets”) and a non-fiction news item will be used to pique curiosity and perhaps motivate students to read more about genetic engineering and it’s tremendous impact on society.

Laboratory exercises will include DNA extraction, comparison of chromosome banding patterns, transformation of bacteria using an engineered plasmid, and callus induction from seeds.

Scope. Pieces may be mixed and matched according to time constraints and laboratory accessibility. Activities are designed to be cost-effective and not to require extensive preparation on the part of the student or teacher. If all components are included, the module may be accomplished over six days. If the novel is omitted and just the short story used, the laboratory exercises may be accomplished in four days.

Chemical Engineering. Chemical engineers utilize chemistry to solve problems for industry. This module focuses primarily on the biochemical aspects of engineering. As science and technology seem to be producing information at astonishing speed, the ‘making it work’ is the job of the chemical engineer. The American Institute of Chemical Engineers (AIChE) has compiled a list of the “10 Greatest Achievements of Chemical Engineering”. The two on the list that pertain to this module are:

1. The Human Reactor – Helping to improve clinical care leading to mechanical wonders such as artificial organs.

2. Wonder Drugs for the Masses – Today’s low price and high volumes of antibiotics owe their existence to the work of chemical engineers.

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When this list was made, the authors would surely have seen the engineering of transgenic organs as science fiction!

To see a summary of each of the ten achievements and to learn more about the field of Chemical Engineering visit: http://www.cems.umn.edu/~aiche_ug/history/h_whatis.html

Chemical Engineering Application and Inspiration. Chemical engineers at Washington State University (WSU) are working on lowering costs in the mass production of desirable proteins. Plant cell suspension cultures are being used as the host to produce recombinant proteins such as GM-CSF which is a human growth factor.

Problem Statement. Genetic engineering has outpaced the comprehension of most citizens. Ethical, legal and moral questions arise as the ‘remotely possible’ becomes probable. Students today will be faced with making decisions about how scientific knowledge is to be used and regulated. An understanding of how our perception of genetic engineering is arrived at is useful to students in determining if their perception has merit. An understanding of the actual principles and technology used to engineer pharmaceuticals, tissues and even new organisms will give students the tools to evaluate new information more critically. By combining fiction, non-fiction, and laboratory experience, students will have the opportunity to integrate literature and science.

The Essential Academic Learning Requirements. When students finish this module they will have achieved many of the items specified by the Washington State “Essential Academic Learning Requirements” (EALRs) and by national standards.

1. The student understands and uses scientific concepts and principles. 2. The student conducts scientific investigations to expand understanding of the natural

world. 3. The student applies science knowledge and skills to solve problems or meet challenges. 4. The student uses effective communication skills and tools to build and demonstrate

understanding of science. 5. The student understands how science knowledge and skills are connected to other subject

areas and real-life situations.

Background Information. According to the National Institute of Health, recombinant DNA technology is defined as: a body of techniques for cutting apart and splicing together different pieces of DNA. When segments of foreign DNA are transferred into another cell or organism, the substance for which they code may be produced along with substances coded for by the native genetic material of the cell or organism. Thus, these cells become "factories" for the production of the protein coded for by the inserted DNA.

“Biologics, which include protein hormones, engineered protein-based vaccines, and monoclonal antibodies, can precisely modify a patient's physiology, often with greater success and fewer side effects than traditional small-molecule drugs or vaccines. Indeed, early biologics—Amgen's (Thousand Oaks, CA) recombinant erythropoietin and Genentech's (S. San Francisco, CA) human growth hormone somatropin—have proven that these drugs can benefit huge numbers of patients

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and generate handsome profits. But biologics are fast becoming victims of their own success, and a looming deficit in biomanufacturing capacity threatens to restrict the expansion of the commercialization of this group of products.

The current standard technology in biomanufacturing, which uses cultured Chinese hamster ovary (CHO) cells in bioreactors, presents major difficulties for companies seeking to scale up. Because nutrients, heat, and gases must diffuse evenly to all cultured cells, the laws of physics set strict limits on the size of bioreactors. Building more bioreactors multiplies costs linearly. A CHO cell–based biomanufacturing plant can cost upwards of $250 million, and an error in estimating demand for, or inaccurately predicting the approval of, a new drug can be incredibly costly. To compound the problem, regulators in the United States and Europe demand that drugs be produced for the market in the same system used to produce them for the final round of clinical trials, so companies have to build facilities for drugs that might not be approved.” (Dove, 2002)

Table 1 shows many of the biotech companies actively pursuing biomanufacturing and the approximate costs.

Dove (2002)

Alan Dove’s article “Uncorking the biomanufacturing bottleneck” can be viewed in the nature biotechnology archives at http://www.nature.com/nbt/ under August 2002. A real fear is that microbes or animal cultures will harbor pathogens. Plant cell cultures do not harbor human pathogens and the productions costs are low but keeping genetically modified pollen from being blown to other plants is worrisome.

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“Plant cell media are composed of simple sugars and salts and are therefore less expensive and complex than mammalian media. Consequently, purification of secreted protein is simpler and more economical. Additionally, plant cell proteins are likely to be safer than those derived from other systems, since plant cell pathogens are not harmful to humans.” (James and Lee, 2001)

Researchers at WSU are using a four-step process to move desirable genes into plant cells.

“The generation of transgenic plant cells involves 4 basic steps (figure 1). The first step is to subclone the gene of interest into a binary Ti plasmid that contains genes for both antibiotic selection and bacterial propagation of the plasmid and for selection of transgenic plant cells. The second step involves the transformation of the bacterial vector, Agrobacterium tumefaciens, with the newly constructed vector. The third step involves the transformation of a tobacco cell line. With A. tumefaciens and selection of transgenic cells, which grow as clumps of cells (calli) on agar plates containing the appropriate antibiotic for selection. The fourth step involves the screening of the individual calli for production of the transgenic protein. A final step involves subcloning the calli cells and selecting for the highest producing lines.” (Magnuson, Wang, James, An, Reeves, and Lee, 2002)

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cDNA

Binary Ti plasmid

Transformation

infection

Agrobacterium tumefaciens

Tobacco cell line: NT-1 cells

callus formation with antibiotic selection

1. Test for protein production Test for mRNA via ELISA production 2. Test for biological function

Fig. 1 illustrates the four basic steps in plant cell transfection. Courtesy of Dr. Nancy Magnuson, Washington State University

Plant suspension cultures are obtained and propagated as calli (undifferentiated cells) which are similar to mammalian stem cells. Stem cells have the remarkable potential to develop into many different cell types in the body. Serving as a sort of repair system for the body, they can theoretically divide without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell. Figure 2 shows how stem cells may be used to repair specific tissues.

Figure 2. The Chemoattractive Hypothesis of Stem-Cell Homing.

Cells with chemokine receptors circulate to various tissues that secrete chemo-attractive molecules. The area of injury secretes chemokines in large amounts, attracting circulating stem cells. (Rosenthal, 2003)

Scientists are currently investigating the potential of various adult stem cells. According to Rosenthal (2003), Table 2 indicates the most common cell types being studied.

Table 2.

Science fiction writers have popularized the suggestion that these cells could someday produce organs for transplantation. Now, it seems, they may not be so “wacky”!

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“Stem cells have been found that can be successfully transplanted from one species to another without immune system rejection, a discovery that could bring stem cell treatments for brain disorders closer.

Researchers from Kansas State University, publishing in the journal Experimental Neurology, report that they have xeno-transplanted umbilical cord matrix stem cells from a pig into the brain of a rat without the rejection of the foreign cells by the rat's immune system.

Umbilical cord matrix stem cells are extracted from a material called Wharton's jelly, a gelatinous connective tissue that helps maintain the umbilical cord's structure and protect its blood vessels.

While they aren't sure why or how, the researchers found that the transplanted cells survived for six weeks undetected, without rejection and without the use of any drugs to suppress an immune response.

It is common for the immune system to reject foreign cells, especially those from other species, and the response poses serious limitations on the success of cell and organ transplants -- especially xeno-transplants.

To counteract immune system rejection, patients are usually put on immunosuppressive therapy. But often, complications arise from immune suppression or from secondary effects of immunosuppressive drugs.

Something about from pig umbilical cord matrix, however, allows them to be ignored by the immune system. And because a subset of the transplanted stem cells respond to the chemical environment of the brain and develop into cells commonly found in the nervous system, they could eventually be used to treat human brain disorders.

"Specifically, the umbilical cord matrix cell source may offer us a basis for treating nervous system disorders like Parkinson's disease," says neuroscientist Mark Weiss.

As evidence from previous studies shows that human umbilical cord matrix cells can differentiate into nervous system tissue, Kansas State researchers are now extending the findings to test human transplant suitability.” (Hunter, 2003)

Materials to obtain before starting the module. One paperback copy of “Chromosome Six” per class involved in the module. You will be tearing it apart so a cheap, well-used copy is preferable. Directions for reading this book in a collaborative, quick manner are included in Appendix A.

One copy per student of “Ethics of the Prophets” found in appendix B.

Bacterial transformation supplies (micropipets, E. coli, plasmid). Any ‘glow in the dark’, ‘green gene’ or x-gal transformation kit will work. I recommend kit IND-9 from Modern Biology, Inc. which sells for about $75.00 per kit. http://www.modernbio.com/ind-9.htm Once you are familiar with the techniques and materials required, you can order by item and save money.

Make student copies of “Comparison of Human and Chimpanzee Chromosomes” found at ENSIweb (Evolution and Nature of Science Institute) for each student. Several hi-liters in

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different colors for student groups of two to four are helpful. http://www.indiana.edu/~ensiweb/ The lesson may be found by clicking ‘The Lessons’, ‘Evolution’, ‘List of Titles’, ‘Comparison of Human and Chimpanzee Chromosomes’. Student pages are in pdf format.

High School teachers within the state of Washington can obtain lab equipment, such as micropipets, through the equipment loan program at Washington State University. Information can be found at this address on the internet http://www.sci.wsu.edu/bio/equiploa.html

Prerequisite Knowledge and Skills. Students should have experience with the idea of sterile technique. Keeping work areas clean and being aware of possible sources of contamination will facilitate the lab work. Students should practice using micropipettors to transfer solutions, streaking agar plates, using pipettes (eye droppers) and handling microcentrifuge tubes. A basic understanding of DNA replication, plasmid structure and protein synthesis is useful but not essential.

Daily Activities

Each day’s activities are designed for 85 minutes periods although the module could be divided into shorter periods.

Day One - Start by introducing the novel (Appendix A) and going through two rotations of reading. This should take about 30-35 minutes. The short story (Appendix B) may be used here if you are omitting the novel.) Discuss scientific terms and procedures mention in the section (10 minutes). Use it to introduce the DNA extraction from Kiwi. (Appendix C) The kiwi extraction takes about 40 minutes.

Day Two – Go through two more rotations of the novel. Each rotation should take about 12 minutes. This would be a good place to discuss the difference between bonobos and chimpanzees. The activity, “Comparison of Human and Chimpanzee Chromosomes” asks students to consider the relationships between humans, chimpanzees and bonobos and takes about 50 minutes. If more information about bonobos would be helpful, I recommend the video from National Geographic, “New Chimpanzees” (1995).

Day Three - Go through two more rotations of the novel. Review/introduce plasmids and DNA structure and function. Practice biotechnology techniques (Appendix D). Set up Callus Induction experiment (Appendix E). This experiment is long-term and can take a month for callus to form. It also is susceptible to contamination by fungi or bacteria. If you choose to include it, it is a good example of plant cells that are similar to mammalian stem cells.

Day Four – Bacterial Transformation (Appendix F) lab IND-9 usually takes the entire 85-minute class period. Students will already have covered approximately 300 pages of the novel at this point. I have included student pages for inserting the pGreen plasmid from Carolina Biological, Inc. into E. coli colonies picked from a petri dish.

Day Five – Go through as many rotations as need in order to finish the novel. Have students finish focus questions and discuss or turn them in. Check on bacterial transformations. Collect

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class data (colony counts) for the bacterial transformations and discuss the results. Depending on which kit (plasmid system) you choose, colonies may need an extra day or two. The Ind.-9 kit from Modern Biology, Inc. usually is ready the next day if you have an incubator.

Day Six – Discuss transfection of plant cells and the direction biomanufacturing is going. See background material for information to include. Researchers use E. coli to design plasmids and then transfer the plasmids from the E. coli to Agrobacterium tumefaciens. A. tumefaciens incorporates the plasmid into its own genomic DNA and then is able to inject its DNA into plant cells. Plant cells expressing the desired protein can then be selected and used in biomanufacturing. This must be done under super-sterile conditions and is not easily done (yet) in the school setting. Students should be able to see the connection between transforming E. coli and the process of transfecting plant cells. This would be a good time to wrap up the module by correcting focus questions and lab conclusions.

Evaluation

A. The novel has a three-page epilogue that does not explain what happened to Kevin Marshall (the researcher responsible for the transgenic bonobos). Have students write an addition to the epilogue or a prologue for a new book, describing his next venture, transfecting plant cells. Ask them to include information about DNA extraction, chromosomes, transformation of bacteria and transgenic organisms.

B. The short story (Appendix B) is a cautionary tale. Ask students to write an essay or letter in response. If the short story is used in addition to the novel, the home group could write using the jigsaw method where each student is responsible for one of the paragraphs in the essay or letter.

C. Give a quiz over the focus questions and lab process.

References

Dove, A., Uncorking the Biomanufacturing Bottleneck, Nature Biotechnology, vol. 20, August 2002, pp. 777-779 or at http://www.nature.com/nbt

Hunter, D., Cross-species Stem Cell Transplantation Holds Promise for Treating Brain Disorders, 6/20/03, http://www.betterhumans.com.

James, E. and Lee, J. The Production of Foreign Proteins from Genetically Modified Plant Cells, Advances in Biochemical Engineering/Biotechnology, vol. 72, 2001, pp. 128-156.

Magnuson, N., Wang, Z., James, E., An, G., Reeves, R., and Lee, J., Plant Cell Culture Expression Systems, Gene Cloning and Expression Technologies, Eaton Pub. 2002, pp. 179-193.

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Rosenthal, N., Prometheus's Vulture and the Stem-Cell Promise, The New England Journal of Medicine, vol 349:267-274, no. 3, July 17, 2003

Schwan, B. Ethics of the Prophets, http://thewritegallery.com/writing/ethics_prophets.html

Acknowledgements

My thanks go to:

Dr. James Lee and Ryan Soderquist for the opportunity to work with transgenic plant cells in their lab at Washington State University.

Dr. Richard Zollars for the opportunity to work with chemical engineers and colleagues during project SWEET at Washington State University and for his support and encouragement.

Dr. Donald C. Orlich of Washington State University for his advice and encouragement in the

writing of this module.

The National Science Foundation for the opportunity to perform unique research and for their support in development of this curriculum.

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Appendix A

“Chromosome Six” – One novel, One week, One copy Objective: Read one copy of a 400-500 page novel, in class, in as little as 4 days! Materials: One paperback copy to be torn up

One copy for teacher reference Focus Questions

Preparations:

1) Divide your classes into groups using your smallest class as the standard. You will need 3 groups of 3, or 4 of 4, 5 of 5, etc. If your smallest class has 26 students, make up 5 groups of 5. The extra student can be paired with another student as a “buddy”. Extra students as buddies also help to fill in if there are absences.

2) This group will be the Home Group. Label each student in a home group with a letter, A, B ,C, D, E.

Example: Group 1 A, B ,C, D, E Group 2 A, B, C, D, E and so on...

3) Using a marker, write the letter “A” in the upper right hand corner, front side only, on the first five pages of text. Paperclip these pages together. Write “B” on the next five pages and clip, then “C” on the next five. Repeat with “D” and “E”. Start over with “A” and continue this process until the entire book is labeled and clipped.

E 49

E 47 E 45

E 43 E 41

A 9 A 7

A 5 A 3

A 1

B 19 B 17

B 15 B 13

B 11

C 29 C 27

C 25 C 23

C 21

A 39 D 37

D 35 D 33

D 31

Sample of “Round 1” Layout

4) Explain to students that they will be reading and discussing the book in class. They should read for understanding, not detail. They will look for answers to the Focus Questions as they’re reading. Students should be able to summarize their page in one sentence. (This takes a bit of practice.)

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Procedures: 1) Start students in their Home Groups, have them move to their Reading Group based on

their letter. All “A” students should sit together, etc. Pass out the reading pages so that each student has one page (front and back) to read. They will have three minutes to read.

2) After 3 minutes, call ‘Time’ and tell them now to go around their table in page order and

summarize their page in 1 sentence. Then, as a group summarize those 10 pages in a few sentences. They will have 3 minutes for this phase. Collect the book pages and collate them.

3) After 3 minutes, they should move back to their Home Groups. They will summarize

their 10 pages for their Home Group with student A going first, then B, etc. This phase also occurs in 3 minutes time. They will now have the main plot for the first 50 pages of the book in only 9 minutes! Students should be allowed 5 minutes to work on the focus questions. You may have individuals work on their own or home groups work together.

4) At this point, the teacher should take a moment to check for comprehension and

understanding of the story and the process. Did they find the answers to the Focus Questions for those pages? Students will be upset with the few that didn’t read well enough or explain well enough to get a focus question. The first day, it might be a good idea to go over the questions as a class. The students not taking their task seriously should now know how to read for understanding and realize that the rest of the group is counting on them.

5) Move back to their Reading Groups and repeat the process with the next 50 pages.

6) The first day will usually cover 2 rotations or the first 100 pages for 5 groups of 5. The

next day they will be able to complete at least 4 rotations. They will become very accomplished at picking out what’s important and summarizing.

** You can figure out how long to spend on this after you get the hang of it on the first day. It should take about half an hour to do the first 2 rotations and then be speedier after that. You can then do as many or few rotations as needed to fit in with the rest of your activities.

Focus questions and answer key follow.

Evaluation Options: 1) Have students write a group essay using the jigsaw method. In a Home Group of 5, each

student is responsible for writing 1 paragraph of a 5-paragraph essay. 2) Give a quiz on the Focus Questions.

3) This novel has a weak ending. Ask students to write an epilogue to the epilogue!

Adapted from “Tearing up the Hot Zone”. An activity presented at 2001 NABT by Sherie Jenkins and Angela Feldbush, West Shore Jr/Sr High School, Melbourne, Florida.

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Chromosome 6 - Focus Questions p. 1-47 1. How is Kevin feeling?

2. What is happening that has brought on Kevin’s feeling?

3. What is Kevin’s office like?

4. What, specifically, does Kevin work on?

5. Where is the research facility located?

6. Why are the GenSys people worried about the Franconi murder?

7. How does Laurie become involved in Kevin’s problem?

8. How does Dr. Raymond Lyons solve the Franconi problem?

9. What tells you that Jack is a ‘risk-taker’?

p. 48-102 10. Where does Kevin go to enlist help? Does he get any?

11. What is GenSys’ business in Cogo?

12. How does Bertram keep track of the ‘creatures’ on Isla Francesca?

13. Kevin is worried about something happening on the island. What?

14. What is Kevin doing to chromosome six?

15. What does he think is happening to the bonobos?

16. What would make you think that Siegfried Spallek is a scary character?

17. What is unusual about the corpse Jack is assigned to autopsy?

18. Why does Raymond visit Dr. Anderson?

p. 103-150 19. What does Kevin finally confess to Candace and Melanie?

20. What do the bonobos seem to be doing that is human-like?

21. Why are Kevin and the women afraid to visit the island?

22. What does Alphonse tell Kevin, Candace and Melanie about the bonobos?

23. What happens when they reach the landing across from the island?

24. How are Jack and Ted going to try to identify the liver tissue in Jack’s

‘floater’?

25. What does Raymond do to try to keep GenSys’ operation secret?

26. What has happened to Kevin and the women after their trip?

27. What do the three decide to do once they are released?

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Chromosome 6 - Focus Questions

p. 151-200 28. How do they get away with their task?

29. What is strange about the specimen of liver tissue Jack looks at?

30. Why is Cindy Carlson abducted?

31. How is Raymond implicated?

32. What surprise do Laurie and Lou have for Jack?

33. Where is Raymond going and why?

p. 201-250 34. What explanation does Bertram give for the smoke coming from the island?

35. How does Kevin make sure they aren’t followed?

36. Did they make it across the bridge? Explain.

37. Why is Jack in trouble with his boss?

38. What does Jack do that alarms the doctors involved with GenSys?

39. Why are Jack and Laurie in danger?

40. How did Kevin, Candace and Melanie explain their second trip to the landing?

41. What is decided over dinner?

42. What does the DQ alpha test tell Ted about the Franconi liver?

43. Why is Jack astonished about the cyclosporin A and FK506 tests?

p. 251-300 44. What does Laurie keep trying to figure out?

45. Why are Raymond, Siegfried and Bertram not worried about Kevin anymore?

46. How does Laurie figure out how Franconi’s body was stolen?

47. How does Lou figure out where Franconi had gone for his transplant?

48. What is a xenograft?

49. How do Franco and Angelo try to scare Laurie and Jack?

p. 301-350 50. Why do Jack and Laurie decide to go to Africa?

51. How do Kevin and the women get to Isla Francesca?

52. What happened to bonobo number sixty?

53. What do the bonobos do with Kevin, Candace and Melanie?

54. Ted tells Jack that the liver would have to be “transgenic”. Explain.

p. 351-400 55. Why does Bertram think the bonobos are killing each other?

56. Who went to Africa with Jack?

57. What happened to Kevin’s escape attempt from the cave?

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Chromosome 6 - Focus Questions

58. How do Jack and his friends get to Cogo?

59. What happens when Jack’s group arrives at the hospital in Cogo?

60. How does Jack figure out what is going on with the livers?

p. 401-456 61. Why are Jack and his friends in serious trouble?

62. Why are Kevin, Candace, and Melanie “rescued”?

63. What are Dave and the men doing to the bonobos?

64. What happens to Kevin and the women when they get back to town?

65. How does Kevin’s group escape?

66. How do they join forces with Jack’s group?

67. Before leaving the area, what does the group decide to do?

68. How do they get the bonobos to cooperate?

69. What did the group have to do to escape the island?

70. Who met Jack’s group at the airport?

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Chromosome 6 - Focus Questions KEY p. 1-47 1. How is Kevin feeling? Anxious, nervous, fearful

2. What is happening that has brought on Kevin’s feeling? An operation

3. What is Kevin’s office like? Hi-tech, futuristic

4. What, specifically, does Kevin work on? Chromosome 6, MHC (major histo-

compatibility complex

5. Where is the research facility located? Cogo, Equatorial Guinea (tropical)

6. Why are the GenSys people worried about the Franconi murder? An autopsy

could cause trouble

7. How does Laurie become involved in Kevin’s problem? She was going to do

the autopsy

8. How does Dr. Raymond Lyons solve the Franconi problem? Calls Dr. Levitz

to contact a New York crime family. They steal the corpse.

9. What tells you that Jack is a ‘risk-taker’? rides his bike fast, been mugged,

lives in a poor neighborhood

p. 48-102 10. Where does Kevin go to enlist help? Bertrom Edwards (veterinarian) Does he

get any? no

11. What is GenSys’ business in Cogo? Animal center, primate testing

12. How does Bertram keep track of the ‘creatures’ on Isla Francesca? Embedded

microchip and satellite tracking

13. Kevin is worried about something happening on the island. What? Bonobos

capable of building fires

14. What is Kevin doing to chromosome six? Putting the short arm of the human

client’s chromosome six on the bonobos’ chromosome six

15. What does he think is happening to the bonobos? They are becoming more

human

16. What would make you think that Siegfried Spallek is a scary character? He has

human skulls on his desk, hunts big game

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17. What is unusual about the corpse Jack is assigned to autopsy? No

heads/hands, sliced up, torso shot to pieces

18. Why does Raymond visit Dr. Anderson? Sign him up to recruit clients

p. 103-150 19. What does Kevin finally confess to Candace and Melanie? His fear that the

bonobos are becoming proto-humans

20. What do the bonobos seem to be doing that is human-like? Vocalizing, fire,

hand gestures, walking upright, aggression

21. Why are Kevin and the women afraid to visit the island? It is a capital offense

22. What does Alphonse tell Kevin, Candace and Melanie about the bonobos?

They fight over food, vocalize, stand, gesture, and that there is a bridge

23. What happens when they reach the landing across from the island? It gets

dark and they find the bridge needs a key. Are fired on by soldiers.

24. How are Jack and Ted going to try to identify the liver tissue in Jack’s

‘floater’? run a DQ alpha test to see if the liver came from a different person.

25. What does Raymond do to try to keep GenSys’ operation secret? Checks up

on other transplant clients and decides to have one ‘disposed of’.

26. What has happened to Kevin and the women after their trip? Thrown in jail

27. What do the three decide to do once they are released? Steal the bridge key

p. 151-200 28. How do they get away with their task? Hide in a refrigerator

29. What is strange about the specimen of liver tissue Jack looks at? no

inflammation

30. Why is Cindy Carlson abducted? She might kill herself and an autopsy would

show her to have had the same type of transplant

31. How is Raymond implicated? The killers take his picture with her corpse

32. What surprise do Laurie and Lou have for Jack? CNN video of Franconi’s

murder match the wounds in Jack’s ‘floater’.

33. Where is Raymond going and why? Africa to pick up the last transplant patient

p. 201-250 34. What explanation does Bertram give for the smoke coming from the island?

Workers clearing paths

35. How does Kevin make sure they aren’t followed? Uses a tunnel to meet the

women

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36. Did they make it across the bridge? no Explain. Soldiers see them and shoot

out the car window

37. Why is Jack in trouble with his boss? He thinks Jack leaked a story to the press

38. What does Jack do that alarms the doctors involved with GenSys? Visits Dr.

Levitz

39. Why are Jack and Laurie in danger? They are getting too close to solving the

Franconi corpse disappearance

40. How did Kevin, Candace and Melanie explain their second trip to the landing?

Melanie said they were looking for the sunglasses she dropped the day before

41. What is decided over dinner? To get to the island by boat

42. What does the DQ alpha test tell Ted about the Franconi liver? matches

43. Why is Jack astonished about the cyclosporin A and FK506 tests? No

immunosuppressive drugs were used

p. 251-300 44. What does Laurie keep trying to figure out? How Franconi’s body was stolen

45.Why are Raymond, Siegfried and Bertram not worried about Kevin anymore?

They think he was scared off and is just hanging out with the women now

46. How does Laurie figure out how Franconi’s body was stolen? Accession #

47. How does Lou figure out where Franconi had gone for his transplant? Follows

the flight plans of the GenSys plane

48. What is a xenograft? Xenotransplantation – ex. Pig heart into a dog

49. How do Franco and Angelo try to scare Laurie and Jack? Beat on Laurie and

kill her cat, try to do the same to Jack but Warren stops them

p. 301-350 50. Why do Jack and Laurie decide to go to Africa? Find out what they can about

GenSys AND to stay away from Franco and Angelo’s people

51. How do Kevin and the women get to Isla Francesca? Boat, and canoe

52. What happened to bonobo number sixty? Was ‘murdered’ by other bonobo

53. What do the bonobos do with Kevin, Candace and Melanie? Capture them and

take them to the caves

54. Ted tells Jack that the liver would have to be “transgenic”. Explain. Most of

the DNA from the liver doesn’t match Franconi but some of it does

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p. 351-400 55. Why does Bertram think the bonobos are killing each other? 2 aren’t moving

and they split into groups earlier

56.Who went to Africa with Jack? Laurie, Warren, Natalie, Esteban

57. What happened to Kevin’s escape attempt from the cave? #1 caught him

58. How do Jack and his friends get to Cogo? boat

59. What happens when Jack’s group arrives at the hospital in Cogo? See Mr.

Winchester and then were chased and finally caught

60. How does Jack figure out what is going on with the livers? He talks to

Rolanda who tells him about moving genes from one chromosome to another

p. 401-456 61. Why are Jack and his friends in serious trouble? Siegfried is going to turn

them over to the Equatoguineans to be executed

62.Why are Kevin, Candace, and Melanie “rescued”? the bonobos are being

rounded up to be moved off the island

63. What are Dave and the men doing to the bonobos? Tranquilized and caged

64. What happens to Kevin and the women when they get back to town? They are

under house arrest and will be given to the Equatoguineans to be executed

65. How does Kevin’s group escape? Give the guards wine

66. How do they join forces with Jack’s group? Break them out of jail

67. Before leaving the area, what does the group decide to do? Free the bonobos

68. How do they get the bonobos to cooperate? Free #1 and use hand signals and

words while freeing the others, send them over the bridge to the jungle

69. What did the group have to do to escape the island? Find the canoe at the

hippo pond

70. Who met Jack’s group at the airport? Lou

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Appendix B

Ethics of the Prophets by Bill Schwan Permission to reprint this story was given to Mari Knutson July, 2004

"Do you remember what you said back then, Ben?" my long time associate Julian asked me, as I admired the small statuette that had been given to me earlier in the day at a small ceremony honoring the one-hundredth anniversary of the legalization of cloning.

"'The results could be staggering. The potential for mankind is just too great to allow politics, soft money or religious zealots to stand in our way.'" It would have been hard for me to forget the quote. It had been the subject of some needlepoint my wife had done years ago and still hung proudly behind the desk in my office. Over the years, the way I had worded things in that address before Congress troubled me, but when placed beside all that had happened in the years since, the angst those words caused me was moot.

"And they were indeed staggering. You're on what, your third heart?" I nodded and said, "Now don't forget that there were plenty of problems to work out, but

given time and a little enlightened thought, work them out we did." The initial problem to overcome had been the funding issue. We had pressed for federal

funding at the start because we didn't expect private industry to jump right into the ethical maelstrom of cloning. But five years after the initial seed money was spent and it became obvious what the technology would be able to achieve, strange bedfellows were seen with increasing regularity. The big tobacco companies worked into the settlement of one of the final federal lawsuits the provision to help fund the cloning of lungs for those affected by use of their products.

Admittedly, it seemed right and even noble for RJR and its kin to fund such research, but in some corners we heard cynical voices accusing the tobacco barons of simply preserving their market. "Of course they're funding cloning research. If a person can just grow a new lung, then they need not fear disease and can perpetuate an irresponsible habit to the eternal joy of stockholders."

Then there was the problem of misunderstanding our motives. The function of the cloning bill had never been to make copies, but rather to set up a guideline for growing a spare organ should the need arise, or to jump start the pancreas of a child with juvenile diabetes. The opposition from the moral high ground had been severe and well organized. I really couldn't fault their arguments, but while I could appreciate what they said, I was also capable of taking the long view. I could see where things had the potential for going whereas they either couldn't see or simply refused to see.

But compromise wins out every time. We came up with a way to market the concept that basically took the average of what people wanted to see done with the technology. No one wanted to see a complete person grown for parts and only the vocal few wanted to ban cloning outright, so we took the mean between the two and the end results made everyone happy. The mean justifies the ends.

Julian chuckled. "The successes were phenomenal in the early years. We grew close to a million kidneys that first year of full-scale production. Hearts were double that. Rheumatoid arthritis treatments brought in a billion-five in the first two years."

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"You know, it's best to think in terms of the amount of pain relieved, not in terms of dollars and cents," I said with a wink.

“Right. Just like choosing to ignore the way the planet went from seven billion people to eight billion in three years time. I know there were accusations aimed at our replacement technology as a chief instigator in the rapid population rise, what with fewer people dying and all, but things always seem to have a way of working themselves out.”

“Yes, the federal government always steps in when necessary. They raised speed limits, repealed the laws requiring air bags and the like, and imposed the death penalty for jaywalking. Interesting the way the death penalty opponents kind of disappeared. If the technology is working for you, then it becomes easy to turn a deaf ear to people who are sacrificed to keep the technology viable. And when automakers couldn't build engines that went as fast as the new speed limits that would pass emissions tests, the government changed the levels of acceptability. That meant that the ozone layer thinned out some more and we had to come up with a way to grow skin in larger quantities faster. Things just worked out better all around."

“And then there are those people who have trouble dealing with a replacement part grown from an embryo. Psychologists and psychiatrists are busier than ever helping folks deal with their guilt feelings, and in the end everyone is happy. The affected eventually feel better about it and live better as a result, the psychiatrists stay busy, and everyone is better off."

At this point, the only problem that remains is the care and maintenance of replacement organs. While the ability to grow a heart for someone is an almost miraculous accomplishment, it still takes five months from stem cell to organ. That means that you either need foreknowledge regarding when your heart muscle is going to cramp up for the last time or you must have a repository of organs on hand, a necessity which is neither cheap nor cost effective. We can usually grow a heart that will be viable for three years after which it must be abandoned and another grown, in my opinion a terrible waste of our resources and the patient's money for the growth and upkeep of an unused organ.

"Well I've got to run. I'm expected to make an appearance at my great great grandson's birthday party tonight, so I'll have to take a raincheck on the racquetball."

"Okay, see you Monday." Half an hour later, I arrived at my great grandson's house. I spent some time chatting with

the parents of the birthday boy and then sat down with the younger man to have some meaningful multi-generational discussion.

"So what are they teaching you in school these days?" I asked, though I already had a fair idea of the curriculum. At age seven, he was probably being introduced to calculus, beginning a second language and hearing about bioethics in health. We had talked to the N.E.A. eighty years earlier about the importance of their role in making the new technologies seem friendly to the future users of those technologies. To that end, a comprehensive plan was developed that set the stage early in the academic career for a life drama that was expected to include growing your own replacement parts at some point. "This week we talked about the kind of work you do, Grampa."

"And just what do you think I do?" "You make hearts for people with bad ones. A fertilized egg divides to a mass of about

two hundred cells, some stem cells are removed, and an organ is grown from those cells. The cells grow in a nurturing machine until it is mature enough to do the job it was meant to do. The teacher told us that this is a perfectly safe and moral way of dealing with illness and not something to give a lot of thought to. It is perfectly ethical and acceptable."

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"And what did you learn?" "Oh, nuthin'!"

Author’s Note This was basically just something I wanted to say to the folks making bold predictions

regarding stem cell research. Apart from any moral implications, I think these folks are courting disaster in the form of problems that no one has even considered. I mean, if you are going to predict the automobile, at least have the decency to predict the traffic jam and smog. The airplane, warn me about jet lag and lost luggage, or you haven't done me any favors.

Focus Questions

• What would you estimate the year to be? Explain.

• How old do you think Ben is? Why?

• What are the two men discussing?

• Summarize three of the unanticipated consequences.

1.

2.

3.

• Describe a product or company that would benefit (not one discussed in the story).

• What is the tone of this story?

• What point does the author make?

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Focus Questions – Possible Answer Key

• What would you estimate the year to be? 2110 Explain. Cloning is not legal yet but may be in the near future.

• • How old do you think Ben is? 150 Why? He has a great, great grandson and was

involved in the early years (100 yrs ago) so was probably an adult then. He has had three heart transplants.

• • What are the two men discussing? How successful the cloning of organs has been and

where the funding came from. • • Summarize three of the unanticipated consequences.

1. tobacco companies contribute funding…new lung = more smokers

2. needing some way to get rid of people…high speed limits

3. needing schools to propagandize

• Describe a product or company that would benefit (not one discussed in the story).

Alcohol companies - livers

• What is the tone of this story? Tongue in cheek, cautionary

• What point does the author make? There are unforeseen consequences to any new technology

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Appendix C Student Handout

DNA Extraction from Kiwifruit Name_________________________Per_____ DNA is present in the cells of all living organisms. This procedure is designed to extract DNA from kiwi in sufficient quantity to be seen and spooled. Some questions to get you thinking about today's lab:

1. One way to purify a molecule is to get rid of everything but that molecule. If we want to isolate DNA from kiwifruit, what do we have to get rid of? Examine this diagram of a plant cell. Fill in the names of the structures you recognize.

2. If you are eating a kiwi or other plant material, how do YOU manage to open the cells? 3. What do you expect DNA to look like?

List of Structures and How to Remove Them in Order to Obtain DNA

Structure Remove how?

Materials per lab team • Zip lock bag • 2 beakers (1 plastic 250ml or bigger, 1 small glass beaker -keep cold or on ice until you need it) • cheese cloth • ice water bath (put some ice and water in a bowl) • extraction solution (detergent and salt in distilled water solution) • 1/2 kiwifruit • cold 95% ethanol • tooth pick • knife (make paper towel ‘cutting board’)

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• 6 centrifuge tubes • microcentrifuge • pipet (eye dropper) • • • • r)

Protocol (Please clean up after yourselves) 1. Get half of a kiwi, peel, chop and put fruit in a ziplock bag. From now on keep the kiwi as

cold as possible to discourage enzyme activity. 2. Add 20-25ml of extraction solution to the ziplock bag. Make sure the bag is closed without much extra air. Mush the kiwi thoroughly but carefully so the bag doesn’t break. Try not to allow foaming. Cool the kiwi mixture in the ice bath for a minute. Then mush the kiwi more. Cool, then mush. Repeat this several times. 3. Filter the mixture through the cheesecloth into the plastic beaker. Throw cheesecloth in trash. Keep kiwi solution cool. 4. Using your pipet, fill 6 microcentrifuge tubes with the and cap tightly. Centrifuge 1 minute. (Keep the rest of the kiwi extraction in case you have trouble.) 5. Pour the supernatant (liquid contents) of all 6 tubes into the icy-cold glass beaker. Pellet of cell debris will stay stuck at the bottom of the tube. 6. Being careful not to shake the beaker, slowly and gently add approximately 1.5cm (1/2 inch) of icy cold 95% ethanol down the side of the beaker to it flows onto the surface of the kiwi solution. 7. Wait 2 minutes. Take a look at the interface of the solutions in your beaker. Did you get DNA? Use a tooth pick to try to spool some DNA. 8. CLEAN UP. Be sure to wash down all surfaces and wash and dry all equipment. Use the toothpick to clean the centrifuge tubes. Check to make sure the tubes are clean before you put them in the drying basket (cap off).

REFLECTION QUESTIONS 1. What does mushing the kiwi do? Why can’t we just start with the extraction solution? 2. What does the extraction solution do to the kiwi? 3. Why do we periodically cool the mixture? 4. When you filtered the kiwi, what was being filtered out (staying in the cheesecloth)?

What was going through the filter? 5. What does the ethanol do? Why do we want it cold? 6. What do you see in the top portion of the liquid after adding the ethanol? 7. Where is DNA found in the cell? What does it look like after extraction? 8. Why was it so difficult for Watson and Crick to determine the structure of DNA? 9. DNA is a chemical common to all living things. What does this suggest about relationships between organisms? 10. Find out what a eukaryotic cell is. Is your kiwi eukaryotic? How do you know this?

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Kiwi-DNA Extraction: Teacher Information

Kiwi DNA Extraction solution recipe. For one liter of the extraction solution, mix 100 ml of shampoo (ex. Suave Daily Clarifying Shampoo, many shampoos will work, but do not use shampoos with conditioner or baby shampoo) and 15 g of table salt (iodized or non-iodized both will work). Add water to make a final volume of 1 liter. Dissolve the salt by stirring slowly to avoid foaming. Need 20-30 ml of solution for each extraction. The detergent dissolves the lipids (fat) in the membranes and the salt protects the DNA from degrading.

Kiwi should be ripe. Need ½ per extraction. If you don’t have a centrifuge, the extraction will still work. Cell debris might cause some discoloration of the DNA. Keep ethanol in the freezer in wash bottles or on ice. Use freezer bags (need to be tougher than sandwich bags or the seeds will pierce) DNA collected is not pure but may be kept in microfuge tubes in a freezer for other experiments. *None of the materials used in the lab exercise are considered hazardous.

Answer Key

DNA in nucleus

Cell wall Cell membrane

E.R.

nucleus

Golgi apparatus

mitochondria chloroplast

Ribosome or lysosome

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List of Structures and How to Remove Them in Order to Obtain DNA

Structure Remove how?

Cell wall Grind, mush (mechanical) Cell membrane

Detergent to dissolve fat in membrane (Extraction fluid)

Nuclear membrane

Detergent

Organelles

Spin to bottom of tube

REFLECTION QUESTIONS

1. What does mushing the kiwi do? Break cell walls Why can’t we just start with the extraction solution? Cell walls won’t dissolve, they are tough 2. What does the extraction solution do to the kiwi? Dissolves membranes 3. Why do we periodically cool the mixture? Keep enzymes from attacking DNA 4. When you filtered the kiwi, what was being filtered out (staying in the cheesecloth)? Seeds,

junk What was going through the filter? Extraction fluid and DNA, broken cells

5. What does the ethanol do? Causes DNA to precipitate Why do we want it cold? Aids precipitation and helps to keep DNA intact…slows enzyme action 6. What do you see in the top portion of the liquid after adding the ethanol? Cloudy material,

DNA 7. Where is DNA found in the cell? Nucleus What does it look like after extraction? Snot,

mucous

8. Why was it so difficult for Watson and Crick to determine the structure of DNA? Looks nothing like its actual structure 9. DNA is a chemical common to all living things. What does this suggest about relationships between organisms? All organisms are related 10. Find out what a eukaryotic cell is. Cells having membranes around organelles and DNA in a nucleus Is your kiwi eukaryotic? yes How do you know this? DNA in nucleus (membrane)

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Appendix D

Genetic Engineering Techniques

Each student needs to practice all three techniques. I set up 2 of each station along a lab table and work with 6 students at a time. I demonstrate all three and then have students rotate between the three types of exercises. It takes about 10 minutes for each group. My other students are working on something else until their turn. Micropipetting. Demonstrate and explain how micropipettes are used. Have students transfer colored (use food coloring) solutions from one microcentrifuge (microfuge) tube to another. Have one micropipette set at 10μl so students can see how small the amount of plasmid will be used and how easy it would be to take too little or too much. Have one micropipette set at a greater amount (250μl or so) so they can feel the difference between the amounts taken up. Students will also gain practice handling microfuge tubes. Opening them with their thumb while holding the tube in the crook of their pointer finger. Have them use an appropriately marked waste receptacle to eject the tip so they get used to looking for the correct disposal containers. Stress sterile technique at all times. Using an ‘eye-dropper’ type pipet. Have a beaker with colored water available. Mark a glass or plastic pipette with the amount you want the student to obtain and move to another beaker. Students have trouble maintaining pressure on the bulb and tend to allow the fluid to move too far up. Have them practice until they can transfer the liquid from one beaker to another without releasing pressure. Plating Bacteria. Have an agar plate, loop and microfuge tube of colored water available. Have students open the microfuge tube, dip the loop, check that the loop has solution across it and then place the loop flat on the agar to streak it back and forth. The lid should be opened slightly but still held over the bottom (sterile technique) The loop should be placed in a disposal container appropriately marked (even if you reuse it for practice). If you use glass beads to spread the bacteria…Have the student use a micropipettor to place 100μl of the colored water onto the center of the plate. The student then places 6 glass bead on the plate and uses a swirling motion to move them over the entire surface. *Not shaking so the beads hit the lid. Continue to remind students they will be working with organisms requiring sterile procedures and careful attention to detail.

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Appendix E Callus Induction Experiment from Plant Seeds

Teacher information Calli are similar to mammalian stem cells in that they are undifferentiated and may be induced to form roots or other plant structures by adding various growth hormones. Calli may be obtained from seeds or leaf punches. Maintaining sterile conditions generally determines the success or failure of tissue culturing. If you have access to a laminar flow hood or at least a tissue culture box such as Carolina’s Transfer Cabinet (19-9713) you will have a better chance of keeping your experiment from contamination. Even if contamination occurs, teachable moments are possible! In order to start fresh callus cultures, the addition of hormones are very important. The purpose of this experiment is to determine optimal conditions of 2,4-D (2,4-dichlorophenoxyacetic acid) and BAP (6-benzylaminopurine) for the induction of callus from several different seeds. Materials are available from Carolina Biological, Inc. Obtain tobacco seeds, carrot seeds and the seeds of any other plants of interest from the store. Buy Murashige and Skoog (MS) solid medium to pour into plates or prepare according to the protocol below. Surface-sterilize seeds by rinsing in 70% ethanol and then soaking in 20% bleach for 15-20 minutes. A section of sterilized pantyhose works well for straining tiny tobacco seeds. Rinse thoroughly with sterile water. Transfer the seeds using sterile forceps onto the plates. Wrap plates with parafilm to retard drying and incubate plates in the dark, optimally at 30oC. Wait for callus formation (can take as long as 1 month. MS Solid Medium (note: growth hormones added after pH adjustment) Reagents. (for 1 liter medium)

1. 4.3 g Murashige and Skoog Salts (Carolina Biologial 19-5701)

2. 30 g Sucrose

3. 10 mL B1 Inositol (4 g myo Inositol and 0.04 g Thiamine HCl in 400 mL distilled water)

4. 3 mL Millers I (12 g KH2PO4 in 200 mL distilled water)

5. 1 Normal KOH

6. Stock of 2,4-D (Under a fume hood add 100 mg of 2,4-Dichlorophenooxyacetic acid to 5

mL 95% Ethanol to dissolve the 2,4-D then add 95 mL distilled water. Cover, remove

from hood and heat gently). Available from Carolina Biological 19-8236.

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7. Stock of BAP (Add 11.25 mg of 6-benzylaminopurine to 2.5 mL of 0.5 M HCl. Heat

slightly to dissolve the BAP. Add 42.5 mL water). Available from Carolina Biological

19-8328.

It is critical that fresh preparations of reagents 3-5 are made periodically. In particular, new 2,4-D must be prepared every 30 days. Procedure.

1) Add 985 mL distilled water to container.

2) Add first four reagents to flask.

3) Mix well and adjust pH to 5.8 with 1 N KOH.

4) Add the appropriate amounts of 2,4-D and BAP. The final 2,4-D conc. is optimally between

1 and 2 mg/L and the final BAP conc. is optimally between 0.25 and 0. 5 mg/L.

5) Dispense medium into a bottle that can be autoclaved. Be sure that some of the liquid

medium is autoclaved in a container that will not receive agar so that the pH of the medium

can be verified after autoclaving.

6) Add 1.3 g Gelrite (Carolina 19-8210) to 500 mL of the liquid medium that will be used to

pour agar plates.

7) Autoclave at 121°C for 15 minutes, after flasks have cooled verify that the medium pH from

a container without agar is approximately 5.2 to 5.3.

Pouring plates. Working in the laminar flow hood, allow the media to cool slightly. Pour medium into petri dishes, then cover and allow solidification, usually 1 hour is sufficient. After plates have cooled to room temperature seal with Parafilm to prevent the medium from dehydrating. Plates may be stored at 4°C for two weeks. Students should periodically check the plates for growth (of any sort) and results can be discussed at that time.

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Appendix F

Bacterial Transformation IND-9 Teacher information- Courtesy of Modern Biology, Inc. The emission of light by living organisms is a fascinating process. The genetic system required for luminescence in the bacterium Photobacterium (Vibiro) fischeri is the lux operon. This operon contains a gene for luciferase (the enzyme that catalyzes the light-emitting reaction) and genes for enzymes which produce the luciferins (which are the substrates for the light-emitting reaction.). In this exercise, students create a luminescent population of bacteria by introducing into E.coli a plasmid that contains this lux operon. The success of the transformation is readily apparent since the E.coli colonies that take up this plasmid glow in the dark as shown below. The simple procedure can be carried out during a single 1-1.5 hour laboratory session. Materials needed that are not provided in the kit include a water bath, incubator and dark room. Teacher preparation includes preparing the nutrient agar-ampicillin plates and preparing the competent cells. This is done by adding E. coli to cold calcium chloride and may be done up to 24 hours before lab but needs to be done at least 10 minutes ahead of time. -Students will then add control plasmid and plasmid lux to two tubes of cells and incubate both on ice for 10 minutes. -They place the tubes in a 37°C water bath for 5 minutes and then add nutrient broth for as long as possible. -The last few minutes of class, the students spread the bacterial suspensions onto agar plates and place in an incubator at 37°C overnight. -View the plates in the light and in the dark the next day or after two days at room temperature. Control Plasmid Plasmid Lux Light Dark The photograph in the bottom panel was taken in total darkness using only the light emitted from the bacteria that were transformed with plasmid lux.

**Have one or two student groups set up a negative control plate with E. coli containing no plasmid. **student responses will depend on lab results.

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Bacterial Transformation: “Glows in the dark” Lab – Student Directions My team needs: 1 beaker, 3/4 full of crushed ice. 1 centrifuge tubes (very small w/ attached cap). 1 ampicillin-nutrient agar plate. 2 sterile pipets (eye droppers). 2 inoculating loops (leave in container until needed so they remain sterile). A clean work table: spray with antiseptic and wipe down with paper towels. 1 pair of gloves for each person who will handle bacterial cultures. Procedure for uptake of DNA by competent E. coli cells:

1. Label one of the tubes C-DNA and the other tube L-DNA. Add an identifying mark to the lid, such as your group number.

2. Put both tubes in your ice bath. 3. Take your tubes (in their ice bath) to the stock table to obtain plasmids. 4. Use the micropipetor (get a clean tip) to add 10μl of the control plasmid to the tube labeled C-DNA. Discard the tip in the waste can. Return the tubes to the ice.

5. Get a new tip to add 10μl of the lux plasmid to the tube labeled L-DNA and again discard the tip in the waste beaker. Return the tubes to the ice.

6. Go to the other stock table and gently tap the tube of competent E. coli cells with the tip of your index finger to ensure that the cells are suspended. Take your sterile pipet.

7. **Be careful not to touch anything after obtaining the bacteria while doing this step and wear gloves.** Using your sterile pipet (eye dropper), add 5 drops of the competent E. coli cells to the C-DNA tube and 5 drops to the L-DNA tube. Discard the pipet in the waste can. Gloves may be removed and kept for later use.

8. Cap tightly and tap the tube with your index finger to mix and place in the ice for 10 minutes.

9. Transfer both tubes to the 37°C water bath for 5 minutes. 10. Use your other sterile pipet to add approx. 0.7 nutrient broth to each tube and put back in the water bath for 35 minutes.(0.7 is mid-way between the .5 mark and the one above it).

Procedure for selection of cells that have taken up the plasmid:

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1. Draw a line down the middle of the bottom of your ampicillin-nutrient plate. Label one side C-DNA and one side L-DNA. Include your group mark and the date. 2. Get one sterile inoculating loop (don’t touch anything other than the bacteria and agar with it once it is removed from the packaging). 3. Wear the gloves and dip the loop into the C-DNA tube and gently wipe it over the correct side of the agar in the C-DNA plate. Repeat 1 more time (for a total of 2 loopsful put on the plate). **Replace the lid on the plate between times.** Discard the loop in one of the waste cans. 4. Get another loop. Again, use sterile technique. Dip the loop into the L-DNA tube and gently wipe it over the correct side of the agar in the L-DNA plate. Repeat 1 more time (for a total of 2 loops-full put on the plate). **Replace the lid on the plate between times.** Discard the loop in one of the waste cans. Discard the DNA tubes in the waste cans too. 5. Use clear tape to tape your plates closed and then set your inoculated plates in the incubator. Remember to invert the plate. 6. Clean up your work area. Spray your table with antiseptic and wipe down with paper towels. Wash your hands thoroughly (lots of scrub action). The plates will be stored in an inverted position, in the dark. They will be ready to view in a completely dark room tomorrow. Allow your eyes to adjust for several minutes in order to see the glowing colonies. **Predict what you think will happen and also answer questions #1-3 on the student analysis page.

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Student Analysis Name_____________________Per____ Pre-Lab: My Predictions Plate Bioluminescent colonies

(yes/no) Explain

NP (no plasmid)

CP (control plasmid pUC18)

Plasmid lux

1. The E. coli you obtained was in an icy-cold calcium chloride solution. Why?

2. Why is an ‘NP’ plate needed?

3. Briefly describe what bacterial transformation is.

Results

My transformation results Plate # of colonies Bioluminescent colonies

(yes/no) NP (no plasmid)

CP (control plasmid pUC18)

Plasmid lux

Class Results Plate # of plates # of colonies

for all plates Bioluminescent colonies (yes/no)

NP (no plasmid)

CP (control plasmid pUC18)

Plasmid lux

4. Why do the cells transformed with pUC18 and with plasmid lux grow in the presence of ampicillin?

5. What is unique about the bacteria transformed with plasmid lux. Explain.

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Appendix G Student Directions for using pGreen plasmid from Carolina Biological, Inc.

1. Mark one sterile 1.5ml microcentrifuge tube “+ plasmid”. Mark another “-plasmid”. Put tubes

on ice. (Make an ice bath by filling a beaker 2/3 full of ice.)

2. Using a micropipettor, add 250 µl of ice-cold calcium chloride to each tube (keep on ice).

3. Use a sterile plastic inoculating loop to transfer several colonies (or about ¼ loop-full) of E. coli to each tube. Stir vigorously so that you are sure the bacteria are well-mixed into the CaCl2. The solution should appear milky white. (Keep on ice.)

4. Transfer 10µl of the pGreen plasmid to the +plasmid tube ONLY. Be sure the tip is in the suspension when you release the plasmids. Keep both tubes on ice for another 10 minutes.

5. While you are waiting, use a sharpie to label your 4 agar plates with your table number, class period and date. These should be written along the outer side of the bottom (agar). Do not write across the bottom or you might not be able to see your bacteria. Label the 4 plates: Amp +, Amp -, No Amp +, No Amp -

6. Heat shock the cells by immediately placing the 2 tubes in a 42°C water bath for 90 seconds.

Stay there and gently agitate the tubes.

7. Return both tubes to the ice for at least one minute. 8. Using a micropipet, add 250 µl of nutrient broth (NB) to each tube and place in a test tube rack at

room temperature. Let sit as long as you can (try for 20 minutes) and still have 10 minutes to plate your bacteria.

9. Pipette 100µl of the + solution onto the appropriate 2 plates and use a sterile loop to spread the

solution around. Repeat, using a new sterile loop for the – solution.

10. Tape the lid on in a couple of places and then when you have all 4 plates done, put them upside down in the appropriate incubator.

**Make your predictions on the analysis page (over).

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