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Student Manual

pGLO Transformation

Lesson 1 Introduction to Transformation

In this lab you will perform a procedure known as genetic transformation. Remember that a gene is a piece of DNA which provides the instructions for making (codes for) a protein. This protein gives an organism a particular trait. Genetic transformation literally means “change caused by genes,” and involves the insertion of a gene into an organism in order to change the organism’s trait. Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of the defective gene that causes the disease. You will use a procedure to transform bacteria with a gene that codes for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria. Green Fluorescent Protein causes the jellyfish to fluoresce and glow in the dark. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. In this activity, you will learn about the process of moving genes from one organism to another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids. Bio-Rad’s unique pGLO plasmid encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on ampillicin plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green under UV light when arabinose is included in the nutrient agar medium. You will be provided with the tools and a protocol for performing genetic transformation. Your task will be to:

1. Do the genetic transformation.
2. Determine the degree of success in your efforts to genetically alter an organism.

LESSON 1STUDENT MANUAL

Lesson 1 Focus Questions

There are many considerations that need to be thought through in the process of planning a scientific laboratory investigation. Below are a few for you to ponder as you take on the challenge of doing a genetic transformation. Since scientific laboratory investigations are designed to get information about a question, our first step might be to formulate a question for this investigation. Consideration 1: Can I Genetically Transform an Organism? Which Organism?

1. To genetically transform an entire organism, you must insert the new gene into every cell in the organism. Which organism is better suited for total genetic transformation—one composed of many cells, or one composed of a single cell?
2. Scientists often want to know if the genetically transformed organism can pass its new traits on to its offspring and future generations. To get this information, which would be a better candidate for your investigation, an organism in which each new generation develops and reproduces quickly, or one which does this more slowly?
3. Safety is another important consideration in choosing an experimental organism. What traits or characteristics should the organism have (or not have) to be sure it will not harm you or the environment?
4. Based on the above considerations, which would be the best choice for a genetic transformation: a bacterium, earthworm, fish, or mouse? Describe your reasoning.

STUDENT MANUAL^ LESSON 1

Consideration 2: How Can I Tell if Cells Have Been Genetically Transformed? Recall that the goal of genetic transformation is to change an organism’s traits, also known as their phenotype. Before any change in the phenotype of an organism can be detected, a thorough examination of its natural (pre-transformation) phenotype must be made. Look at the colonies of E. coli on your starter plates. List all observable traits or characteristics that can be described: The following pre-transformation observations of E. coli might provide baseline data to make reference to when attempting to determine if any genetic transformation has occurred. a) Number of colonies b) Size of : 1) the largest colony

2. the smallest colony
3. the majority of colonies c) Color of the colonies d) Distribution of the colonies on the plate e) Visible appearance when viewed with ultraviolet (UV) light f) The ability of the cells to live and reproduce in the presence of an antibiotic such as ampicillin

1. Describe how you could use two LB/agar plates, some E. coli and some ampicillin to determine how E. coli cells are affected by ampicillin.
2. What would you expect your experimental results to indicate about the effect of ampicillin on the E. coli cells?

LESSON 1STUDENT MANUAL

Consideration 3: The Genes Genetic transformation involves the insertion of some new DNA into the E. coli cells. In addition to one large chromosome, bacteria often contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for more than one trait. Scientists use a process called genetic engineering to insert genes coding for new traits into a plasmid. In this case, the pGLO plasmid has been genetically engineered to carry the GFP gene which codes for the green fluorescent protein, GFP, and a gene ( bla ) that codes for a protein that gives the bacteria resistance to an antibiotic. The genetically engineered plasmid can then be used to genetically transform bacteria to give them this new trait. Consideration 4: The Act of Transformation This transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly acquired genes. To move the pGLO plasmid DNA through the cell membrane you will:

1. Use a transformation solution containing CaCl 2 (calcium chloride).
2. Carry out a procedure referred to as heat shock. For transformed cells to grow in the presence of ampicillin you must:
3. Provide them with nutrients and a short incubation period to begin expressing their newly acquired genes. pGLO plasmid DNA GFP Flagellum Pore Cell wall Beta-lactamase Bacterial^ (antibiotic resistance) chromosomal DNA

STUDENT MANUAL^ LESSON 1

Lesson 2 Transformation Laboratory

Workstation (! ) Checklist

Your workstation: Materials and supplies that should be present at your workstation prior to beginning this lab are listed below. Student workstation Material Quantity (! ) E. coli starter plate 1 " Poured agar plates (1 LB, 2 LB/amp, 1 LB/amp/ara) 4 " Transformation solution 1 " LB nutrient broth 1 " Inoculation loops 7 (1 pk of 10) " Pipets 5 " Foam microcentrifuge tube holder/float 1 " Container (such as foam cup) full of crushed ice (not cubed ice) 1 o Marking pen 1 " Copy of Quick Guide 1 " Microcentrifuge tubes 2 " Common workstation. A list of materials, supplies, and equipment that should be present at a common location to be accessed by your team is also listed below. Material Quantity Rehydrated pGLO plasmid 1 vial " 42°C water bath and thermometer 1 " UV Light 1 " 37°C incubator 1 " (optional, see General Laboratory Skills–Incubation) 2–20 μl adjustable volume micropipets 1 " 2–20 μl micropipet tips 1 "

LESSON 2STUDENT MANUAL

Transformation Procedure

1. Label one closed micro test tube +pGLO and another -pGLO. Label both tubes with your group’s name. Place them in the foam tube rack.
2. Open the tubes and, using a sterile transfer pipet, transfer 250 μl of transformation solution (CaCl 2 ) into each tube. +pGLO +pGLO -pGLO -pGLO Transformation Solution 250 μl

STUDENT MANUAL^ LESSON 2

3. Place the tubes on ice.
4. Use a sterile loop to pick up 2–4 large colonies of bacteria from your starter plate. Select starter colonies that are "fat" (ie: 1–2 mm in diameter). It is important to take individual colonies (not a swab of bacteria from the dense portion of the plate), since the bacteria must be actively growing to achieve high transforation efficiency. Choose only bacterial colonies that are uniformly circular with smooth edges. Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the -pGLO tube.
5. Examine the pGLO DNA solution with the UV lamp. Note your observations. Immerse a new sterile loop into the pGLO plasmid DNA stock tube. Withdraw a loopful. There should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring for blowing soap bubbles. Mix the loopful into the cell suspension of the +pGLO tube. Optionally, pipet 10 μl of pGLO plasmid into the +pGLO tube & mix. Do not add plasmid DNA to the -pGLO tube. Close both the +pGLO and -pGLO tubes and return them to the rack on ice. +pGLO Ice (+pGLO) pGLO Plasmid DNA (+pGLO) (-pGLO) (-pGLO) +pGLO

LESSON 2 STUDENT MANUAL

6. Incubate the tubes on ice for 10 min. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the ice.
7. While the tubes are sitting on ice, label your four LB nutrient agar plates on the bottom (not the lid) as follows:

• Label one LB/amp plate: + pGLO
• Label the LB/amp/ara plate: + pGLO
• Label the other LB/amp plate: - pGLO
• Label the LB plate: - pGLO

8. Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42oC, for exactly 50 sec. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the warm water. Double-check the temperature of the water bath with two thermometers to ensure accuracy. When the 50 sec are done, place both tubes back on ice. For the best transformation results, the transfer from the ice (0°C) to 42°C and then back to the ice must be rapid. Incubate tubes on ice for 2 min. Water bath L B / a m p pGLO L B / am p / a r a pGLO L B / a m p pGLO L B pGLO Rack Ice 42°C for 50 sec Ice Ice

STUDENT MANUAL^ LESSON 2

9. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 μl of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 min at room temperature.
10. Gently flick the closed tubes with your finger to mix and resuspend the bacteria. Using a new sterile pipet for each tube, pipet 100 μl of the transformation and control suspensions onto the appropriate nutrient agar plates. 100 μl Transformation plates Control plates **L B

• p**

G

O

L

L B / a m p

- p

G

O

L

L B / a m p

+ p

G

O

L

L B / a m p /^ a^ r^ a

+ p

G

O

L

LB broth 250 μl +pGLO +pGLO -pGLO -pGLO

LESSON 2 STUDENT MANUAL

11. Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the LB nutrient agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. DO NOT PRESS TOO DEEP INTO THE AGAR. Uncover one plate at a time and re-cover immediately after spreading the suspension of cells. This will minimize contamination.
12. Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack and place the stack of plates upside down in the 37°C incubator until the next day. The plates are inverted to prevent condensation on the lid which may drip onto the culture and interfere with your results. **L B

• p**

G

O

L

L B / a m p

- p

G

O

L

L (^) B / a m p

+ p

G

O

L L

B / a m p / a r a

+ p

G

O

L

STUDENT MANUAL^ LESSON 2

Lesson 2 Review Questions Name ___________________

Before collecting data and analyzing your results answer the following questions.

1. On which of the plates would you expect to find bacteria most like the original non-transformed E. coli colonies you initially observed? Explain your predictions.
2. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions.
3. Which plates should be compared to determine if any genetic transformation has occurred? Why?
4. What is meant by a control plate? What purpose does a control serve?

LESSON 2STUDENT MANUAL

Lesson 3 Data Collection and Analysis

A. Data Collection Observe the results you obtained from the transformation lab under normal room lighting. Then turn out the lights and hold the ultraviolet light over the plates. Alternatively the protocol can incorporate digital documentation of the plates with Vernier’s Blue Digital BioImaging System (Appendix E).

1. Carefully observe and draw what you see on each of the four plates. Put your drawings in the data table below. Record your data to allow you to compare observations of the “ + pGLO ” cells with your observations for the non-transformed E. coli. Write down the following observations for each plate.
2. How much bacterial growth do you see on each plate, relatively speaking?
3. What color are the bacteria?
4. How many bacterial colonies are on each plate (count the spots you see). Observations +pGLO LB/amp +pGLO LB/amp/ara Observations -pGLO LB/amp -pGLO LB Control plates Transformation plates

STUDENT MANUAL LESSON 3

B. Analysis of Results The goal of data analysis for this investigation is to determine if genetic transformation has occurred.

1. Which of the traits that you originally observed for E. coli did not seem to become altered? In the space below list these untransformed traits and how you arrived at this analysis for each trait listed. Original trait Analysis of observations
2. Of the E. coli traits you originally noted, which seem now to be significantly different after performing the transformation procedure? List those traits below and describe the changes that you observed. New trait Observed change
3. If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, then what might be inferred about the other genes on the plasmid that you used in your transformation procedure?
4. From the results that you obtained, how could you prove that the changes that occurred were due to the procedure that you performed?

LESSON 3STUDENT MANUAL

Lesson 3 Review Questions Name _____________________

What’s Glowing? If a fluorescent green color is observed in the E. coli colonies then a new question might well be raised, “What are the two possible sources of fluorescence within the colonies when exposed to UV light?” Explain:

1. Recall what you observed when you shined the UV light onto a sample of original pGLO plasmid DNA and describe your observations.
2. Which of the two possible sources of the fluorescence can now be eliminated?
3. What does this observation indicate about the source of the fluorescence?
4. Describe the evidence that indicates whether your attempt at performing a genetic transformation was successful or not successful.

STUDENT MANUAL LESSON 3

Lesson 3 Review Questions Name ____________________

The Interaction between Genes and Environment Look again at your four plates. Do you observe some E. coli growing on the LB plate that does not contain ampicillin or arabinose?

1. From your results, can you tell if these bacteria are ampicillin resistant by looking at them on the LB plate? Explain your answer.
2. How would you change the bacteria’s environment—the plate they are growing on—to best tell if they are ampicillin resistant?
3. Very often an organism’s traits are caused by a combination of its genes and its environment. Think about the green color you saw in the genetically transformed bacteria: a. What two factors must be present in the bacteria’s environment for you to see the green color? (Hint: one factor is in the plate and the other factor is in how you look at the bacteria). b. What do you think each of the two environmental factors you listed above are doing to cause the genetically transformed bacteria to turn green? c. What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions?

LESSON 3STUDENT MANUAL

Lesson 4 Extension Activity: Calculate Transformation Efficiency

Your next task in this investigation will be to learn how to determine the extent to which you genetically transformed E. coli cells. This quantitative measurement is referred to as the transformation efficiency. In many experiments, it is important to genetically transform as many cells as possible. For example, in some types of gene therapy, cells are collected from the patient, transformed in the laboratory, and then put back into the patient. The more cells that are transformed to produce the needed protein, the more likely that the therapy will work. The transformation efficiency is calculated to help scientists determine how well the transformation is working. The Task You are about to calculate the transformation efficiency, which gives you an indication of how effective you were in getting DNA molecules into bacterial cells. Transformation efficiency is a number. It represents the total number of bacterial cells that express the green protein, divided by the amount of DNA used in the experiment. (It tells us the total number of bacterial cells transformed by one microgram of DNA.) The transformation efficiency is calculated using the following formula: Transformation efficiency = Total number of colonies growing on the agar plate Amount of DNA spread on the agar plate (in μg) Therefore, before you can calculate the efficiency of your transformation, you will need two pieces of information: (1) The total number of green fluorescent colonies growing on your LB/amp/ara plate. (2) The total amount of pGLO plasmid DNA in the bacterial cells spread on the LB/amp/ara plate.

STUDENT MANUAL LESSON 4

1. Determining the Total Number of Green Fluorescent Cells Place your LB/amp/ara plate near a UV light. Each colony on the plate can be assumed to be derived from a single cell. As individual cells reproduce, more and more cells are formed and develop into what is termed a colony. The most direct way to determine the total number of bacteria that were transformed with the pGLO plasmid is to count the colonies on the plate. Enter that number here ➔ 2. Determining the Amount of pGLO DNA in the Bacterial Cells Spread on the LB/amp/ara Plate We need two pieces of information to find out the amount of pGLO DNA in the bacterial cells spread on the LB/amp/ara plate in this experiment. (a) What was the total amount of DNA we began the experiment with, and (b) What fraction of the DNA (in the bacteria) actually got spread onto the LB/amp/ara plates. Once you calculate this data, you will multiply the total amount of pGLO DNA used in this experiment by the fraction of DNA you spread on the LB/amp/ara plate. This will tell you the amount of pGLO DNA in the bacterial cells that were spread on the LB/amp/ara plate. a. Determining the Total Amount of pGLO plasmid DNA The total amount of DNA we began with is equal to the product of the concentration and the total volume used, or (DNA in μg) = (concentration of DNA in μg/μl) x (volume of DNA in μl) In this experiment you used 10 μl of pGLO at concentration of 0.08 μg/μl. This means that each microliter of solution contained 0.08 μg of pGLO DNA. Calculate the total amount of DNA used in this experiment. Enter that number here ➔ How will you use this piece of information? Total number of colonies = _______ Total amount of pGLO DNA (μg) used in this experiment = ___________________________

LESSON 4 STUDENT MANUAL

b. Determining the fraction of pGLO plasmid DNA (in the bacteria) that actually got spread onto the LB/amp/ara plate: Since not all the DNA you added to the bacterial cells will be transferred to the agar plate, you need to find out what fraction of the DNA was actually spread onto the LB/amp/ara plate. To do this, divide the volume of DNA you spread on the LB/amp/ara plate by the total volume of liquid in the test tube containing the DNA. A formula for this statement is Volume spread on LB/amp plate (μl) Total sample volume in test tube (μl) You spread 100 μl of cells containing DNA from a test tube containing a total volume of 510 μl of solution. Do you remember why there is 510 μl total solution? Look in the laboratory procedure and locate all the steps where you added liquid to the reaction tube. Add the volumes. Use the above formula to calculate the fraction of pGLO plasmid DNA you spread on the LB/amp/ara plate. Enter that number here ➔

• How will you use this piece of information? So, how many micrograms of pGLO DNA did you spread on the LB/amp/ara plates? To answer this question, you will need to multiply the total amount of pGLO DNA used in this experiment by the fraction of pGLO DNA you spread on the LB/amp/ara plate. pGLO DNA spread in μg = Total amount of DNA used in μg x fraction of DNA used Enter that number here ➔
• What will this number tell you? Fraction of DNA used = Fraction of DNA =

---

pGLO DNA spread (μg) =

---

STUDENT MANUAL LESSON 4

Look at all your calculations above. Decide which of the numbers you calculated belong in the table below. Fill in the following table. Now use the data in the table to calculate the efficiency of the pGLO transformation Transformation efficiency = Total number of colonies growing on the agar plate Amount of DNA spread on the agar plate (in μg) Enter that number here ➔ Analysis Transformation efficiency calculations result in very large numbers. Scientists often use a mathematical shorthand referred to as scientific notation. For example, if the calculated transformation efficiency is 1,000 bacteria/μg of DNA, they often report this number as: 103 transformants/μg (10^3 is another way of saying 10 x 10 x 10 or 1,000)

• How would scientists report 10,000 transformants/μg in scientific notation? Carrying this idea a little farther, suppose scientists calculated an efficiency of 5, bacteria/μg of DNA. This would be reported as: 5 x 10^3 transformants/μg (5 x 1,000)
• How would scientists report 40,000 transformants/μg in scientific notation? Number of colonies on LB/amp/ara plate = Micrograms of pGLO DNA spread on the plates Transformation efficiency = _____ transformants/μg

LESSON 4 STUDENT MANUAL

One final example: If 2,600 transformants/μg were calculated, then the scientific notation for this number would be: 2.6 x 10^3 transformants/μg (2.6 x 1,000) Similarly: 5,600 = 5.6 x 10^3 271,000 = 2.71 x 10^5 2,420,000 = 2.42 x 10^6

• How would scientists report 960,000 transformants/μg in scientific notation?
• Report your calculated transformation efficiency in scientific notation.
• Use a sentence or two to explain what your calculation of transformation efficiency means. Biotechnologists are in general agreement that the transformation protocol that you have just completed generally has a transformation efficiency of between 8.0 x 10^2 and 7.0 x 10^3 transformants per microgram of DNA.
• How does your transformation efficiency compare with the above?
• In the table below, report the transformation efficiency of several of the teams in the class. Team Efficiency
• How does your transformation efficiency compare with theirs?

STUDENT MANUAL LESSON 4

• Calculate the transformation efficiency of the following experiment using the information and the results listed below. DNA plasmid concentration: 0.08 μg/μl 250 μl CaCl 2 transformation solution 10 μl pGLO plasmid solution 250 μl LB broth 100 μl cells spread on agar 227 colonies of transformants Fill in the following chart and show your calculations to your teacher:
• Extra Credit Challenge: If a particular experiment were known to have a transformation efficiency of 3 x 10^3 bacteria/μg of DNA, how many transformant colonies would be expected to grow on the LB/amp/ara plate? You can assume that the concentration of DNA and fraction of cells spread on the LB agar are the same as that of the pGLO laboratory. Number of colonies on LB/amp/ara plate = Micrograms of DNA spread on the plates = Transformation efficiency =

LESSON 4STUDENT MANUAL

Appendix A

Historical Links to Biotechnology

Biological transformation has had an interesting history. In 1928, Frederick Griffith, a London physician working in a pathology laboratory, conducted an experiment that he would never be able to fully interpret as long as he lived. Griffith permanently changed (transformed) a safe, nonpathogenic bacterial strain of pneumococcus into a deadly pathogenic strain. He accomplished this amazing change in the bacteria by treating the safe bacteria with heat-killed deadly bacteria. In this mixture of the two bacterial strains there were no living, virulent bacteria, but the mixture killed the mice it was injected into. He repeated the experiment many times, always with the same results. He and many of his colleagues were very perplexed. What transformed safe bacteria into the deadly killers? Many years later, this would come to be known as the first recorded case of biological transformation conducted in a laboratory, and no one could explain it. Griffith did not know of DNA, but knew the transformation was inheritable. As any single point in history can be, Griffith’s experiments in transformation can be seen as the birth of analytical genetic manipulation that has led to recombinant DNA and biotechnology, and the prospects for human gene manipulation. In 1944, sixteen years after Griffith’s experiment, a research group at Rockefeller Institute, led by Oswald T. Avery, published a paper that came directly from the work of Griffith. “What is the substance responsible?” Avery would ask his coworkers. Working with the same strains of pneumonia-causing bacteria, Avery and his coworkers provided a rigorous answer to that question. They proved that the substance is DNA, and that biological transformation is produced when cells take up and express foreign DNA. Although it took many years for credit to be given to Avery, today he is universally acknowledged for this fundamental advance in biological knowledge. Building upon the work of Avery and others, Douglas Hanahan developed the technique of colony transformation used in this investigation.1, 2 Historical Context Genetic Transformation 1865—Gregor Johann Mendel: Mendel presented his findings describing the principles by which genetic traits are passed from parent to offspring. From his work the concept of the gene as the basic unit of heredity was derived. 1900—Carl Correns, Hugo De Vries, Erich Tschermak: Plant geneticists conducting inheritance studies uncovered that their work was essentially a duplicate of work performed nearly four decades earlier by an unknown Austrian Augustinian monk, Gregor Johann Mendel, who studied peas. 1928—Frederick Griffith: Griffith transformed nonpathogenic Diplococcus pneumonia into pathogenic bacteria using heat-killed virulent bacteria. He suggested that the transforming factor had something to do with the polysaccharide capsule synthesis. Griffith did not know of DNA, but knew the transformation was inheritable. Griffith’s experiments in transformation can be seen as the birth of analytical genetic manipulation that has led to recombinant DNA technology and the prospects for human gene manipulation.

APPENDIX A

1944—Oswald Avery, Colin MacLeod: Avery and his colleagues announced that they had isolated the transforming factor to a high purity, and it was DNA. Since this classic experiment in molecular genetics, transformation, conjugation (bacterial mating), and transduction (viral DNA transfer) have been used to transfer genes between species of bacteria, Drosophila , mice, plants and animals, mammalian cells in culture, and for human gene therapy. 1952—Alfred Hershey, Martha Chase: Hershey and Chase used radioisotopes of sulfur and phosphorus, and bacteriophage T2 to show conclusively that DNA was the information molecule of heredity. Along with the work of Avery, MacLeod, and McCarty, the Hershey/Chase experiment sealed the understanding that DNA was the transforming material and the information molecule of heredity. 1972—Paul Berg, Janet Mertz: Berg used the newly discovered endonuclease enzyme, Eco RI, to cut SV40 DNA and bacteriophage P22 DNA, and then used terminal transferase enzyme and DNA ligase to rejoin these separate pieces into one piece of DNA. Creation of the first recombinant DNA molecule was the beginning of the age of biotechnology. The new molecule was not placed inside a mammalian cell because of concerns in the scientific community regarding genetic transfers. 1973—Herbert Boyer, Stanley Cohen, Annie Chang: Berg, Boyer, and Cohen used Eco RI to isolate an intact gene for kanamycin resistance. Boyer, Cohen, and Chang spliced the kanamycin resistance gene into an Eco RI cut plasmid that already contained tetracycline resistance and produced a recombinant bacterial plasmid molecule with dual antibiotic resistance. They then transformed E. coli with this engineered plasmid. 1977—Genentech, Inc.: The first product of genetic engineering, the gene for human somatostatin (human growth hormone-releasing inhibitory factor), was expressed in bacteria and announced by Genentech. 1980—J. W. Gordon, Frank Ruddle: Gordon and Ruddle successfully microinjected normal genes into mouse germ-line cells. 1982—Richard Palmiter, Ralph Brinster: Palmiter and Brinster microinjected the gene for rat growth hormone into mice embryos. This was the first genetic germ-line “cure” reported in a mammal. The recipient mouse was called “little” because it suffered from a form of congenital dwarfism. 1988—Steven Rosenberg: Rosenberg and his colleagues were given approval to perform the first gene transfer experiment in human patients suffering from metastatic melanoma. This experiment represented genetic tracking with the marker gene NeoR and not gene therapy. 1990—W. French Anderson, Michael Blaese, Kenneth Culver: At 12:52 p.m. on Friday, September 14, 1990 at the National Cancer Institute, a four year old girl, Ashanthi De Silva from Cleveland, Ohio, became the first human gene-therapy patient. She was infused with her own white blood cells carrying the corrected version of the adenosine deaminase (ADA) gene. Drs. Anderson, Blaese and Culver did not expect meaningful results from the experiment for about 1 year. A second girl, Cynthia Cutshall, was similarly injected in 1990. Reports in June 1993 showed the two girls with smiles and childish energy, playing in a school yard. Both girls’ immune systems were working effectively. 1994—Other gene therapy candidates include sickle cell anemia, hemophilia, diabetes, cancer, and heart disease patients. Germ line gene therapy is debated during meeting of the Recombinant DNA Advisory Committee. By 1996 a growing number of proposals await review by the Human Gene Therapy Subcommittee of the Recombinant DNA Advisory Committee.

APPENDIX A

1995—Led by J. Craig Venter, a group at The Institute for Genomic Research (TIGR) in Maryland, published the full gene sequence of the bacterium Hemophilus influenzae, a landmark in microbiological research as the first free-living organism whose genetic “blueprint” was decoded. 1996—A multinational collaboration including more than 100 laboratories from Europe, USA, Canada, and Japan was the first to unravel the entire genome sequence of a eukaryote, the yeast Saccharomyces cerevisiae. S. cerevisiae is a commercially significant yeast commonly used in baking and in fermentation of alcoholic beverages and is widely used in the laboratory as a model organism for understanding cellular and molecular processes of eukaryotes. 1997—Scientists led by Ian Wilmut at Scotland’s Roslin Institute reported the successful cloning of a sheep, named Dolly, from the cell of an adult udder cell. The cloning of Dolly sparked international debate about ethical and moral issues concerning cloning. Subsequently, scientists at Scotland’s Roslin Institute, in collaboration with Scotland- based PPL Therapeutics, successfully cloned two genetically modified lambs, named Polly and Molly, that were genetically modified with a human gene so that their milk contained a protein called factor IX, a blood-clotting protein that can be extracted and used in treating human hemophilia. 1998—Over 99% of the genome sequence of the first multicellular organism, the tiny roundworm Caenorhabditis elegans , was reported. Although C. elegans is a primitive organism, it shares many of its essential genetic and biological characteristics with humans and may help scientists identify and characterize the genes involved in human biology and disease. Scientists produced a detailed and accurate physical map, or location, for most of the 30,000 known human genes, a milestone for the Human Genome Project. 2000—A team led by Ingo Potrykus of the Swiss Federal Institute of Technology in Zurich and Peter Beyer of the University of Freiburg in Germany reported the creation of genetically modified rice called “golden rice”, which can produce large amounts of beta-carotene, a substance that human beings can turn into Vitamin A. “Golden rice” could alleviate blindness caused by vitamin A deficiency in millions of poverty-stricken people around the world. The genome sequence of the fruit fly Drosophila melanogaster was published through a collaboration between a private company, Celera Genomics, and researchers worldwide studying the fruit fly. D. melanogaster , a model widely used in the laboratory, is the largest animal so far to have its genetic code deciphered. A rough draft of the human genome was completed by a team of 16 international institutions that form the Human Genome Sequencing Consortium. Researchers at Celera Genomics also announced completion of their ‘first assembly’ of the genome.

APPENDIX A

2001—On February 12, 2001, Celera Genomics and the International Human Genome Sequencing Consortium jointly announced the publishing of the nearly complete sequence of the human genome - the genetic “blueprint” for a human being. This accomplishment took the international team almost twenty years and involved the collaboration of thousands of scientists from around the world. Celera Genomics reported completing the work in approximately nine months. The two groups differed in their estimates for the number of genes in the human genome, but the range predicted by both groups, between 25,000 and 40,000 genes, is far fewer than the previous estimate of 100,000 genes. This unexpected finding suggested that an organism as complex as a human being can be made of so few genes, only twice as many as found in the worm C. elegans or the fly D. melanogaster. The unveiling of the full sequence of the human genome makes it possible for researchers all over the world to begin developing treatments for many diseases. President George Bush decided that only experiments involving the existing 64 embryonic stem cell lines would be eligible for possible federal funding. The president’s decision was disappointing to many scientists who hoped to use embryonic stem cells to develop treatments for many ailments. Advanced Cell Technology, a small company in Massachusetts, announced that it had successfully cloned human embryos for the purpose of extracting their stem cells. This method could ultimately be used to treat patients with a variety of diseases by making replacement cells, such as nerve and muscle cells, which can be transplanted back into same person without the risk of being rejected by the body. PPL Therapeutics, the company that helped to clone Dolly the sheep, announced that it had cloned five genetically modified piglets with an inactivated, or “knocked out”, gene that would make their organs much less likely to be rejected when transplanted into a human recipient. The success of PPL Therapeutics brings hope to the thousands of people who are waiting to receive donated organs such as hearts, lungs, kidneys, and livers. 2002—Dolly the sheep, the first mammal to be cloned from an adult cell, developed arthritis at a relatively early age of five years. It is not clear whether Dolly’s condition was the result of a genetic defect caused by cloning, or whether it was a mere coincidence. The news has renewed debates on whether cloned animals are susceptible to premature aging and health problems and has also been a setback for those who argue that cloning can be used to generate a supply of organs to help patients on the transplant list. 2008—Discovery and landmark developmental uses of GFP wins the Nobel Prize in Chemistry. Osamu Shimonura was the first to isolate GFP and found that it had fluorescent properties when exposed to UV light. Martin Chalfie used GFP as a luminous genetic tag. Roger Y. Tsien uncovered GFP's fluorescent mechanism.

APPENDIX A