Lab manual pGLO Transformation, Papers of Molecular biology

Download Lab manual pGLO Transformation and more Papers Molecular biology in PDF only on Docsity! 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. 32 STUDENT MANUAL LESSON 1 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. 33 ST UD EN T MA NU AL LE SS ON 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 " 36 STUDENT MANUAL LESSON 2 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 (CaCl2) into each tube. 37 + p G L O + p G L O -p G L O -p G L O Transformation Solution 250 µl ST UD EN T MA NU AL LE SS ON 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. 38 + p G L O Ice (+pGLO) pGLO Plasmid DNA (+pGLO) (-pGLO) (-pGLO) + p G L O STUDENT MANUAL LESSON 2 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. 41 L B - pG O L LB/am p - pG O L LB/am p +pG O L LB/amp/ara +pG O L ST UD EN T MA NU AL LE SS ON 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? 42 STUDENT MANUAL LESSON 2 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 43 Co nt ro l p lat es Tr an sf or m at io n pl at es ST UD EN T MA NU AL LE SS ON 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? 46 STUDENT MANUAL LESSON 3 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. 47 ST UD EN T MA NU AL LE SS ON 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? 48 Total number of colonies = _______ Total amount of pGLO DNA (µg) used in this experiment = ___________________________ STUDENT MANUAL LESSON 4 One final example: If 2,600 transformants/µg were calculated, then the scientific notation for this number would be: 2.6 x 103 transformants/µg (2.6 x 1,000) Similarly: 5,600 = 5.6 x 103 271,000 = 2.71 x 105 2,420,000 = 2.42 x 106 • 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 102 and 7.0 x 103 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? 51 ST UD EN T MA NU AL LE SS ON 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 CaCl2 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 103 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 = 52 STUDENT MANUAL LESSON 4 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. 53 AP PE ND IX 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. 56 APPENDIX A Appendix B Glossary of Terms Agar A gelatinous substance derived from seaweed. Provides a solid matrix to support bacterial growth. Contains nutrient mixture of carbohydrates, amino acids, nucleotides, salts, and vitamins. Antibiotic Selection Use of an antibiotic to select bacteria containing the DNA of interest. The pGLO plasmid DNA contains the gene for beta-lactamase that provides resistance to the antibiotic ampicillin. Once bacteria are transformed with the pGLO plasmid, they begin producing and secreting beta-lactamase protein. Secreted beta-lactamase breaks down ampicillin, rendering the antibiotic harmless to the bacterial host. Only bacteria containing the pGLO plasmid can grow and form colonies in nutrient medium containing ampicillin, while untransformed cells that have not taken up the pGLO plasmid cannot grow on the ampicillin selection plates. Arabinose A carbohydrate isolated from plants that is normally used as source of food by bacteria. In this experiment, arabinose initiates transcription of the GFP gene resulting in fluorescent green cells under UV light. Beta-Lactamase Beta-lactamase is a protein that provides resistance to the antibiotic ampicillin. The beta-lactamase protein is produced and secreted by bacteria that have been transformed with a plasmid containing the gene for beta-lactamase. The secreted beta-lactamase inactivates the ampicillin present in the LB nutrient agar, which allows for bacterial growth and expression of newly acquired genes also contained on the plasmid, such as GFP. Biotechnology Applying biology in the real world by the specific manipulation of living organisms, especially at the genetic level, to produce potentially beneficial products. Cloning Cloning is the process of generating virtually endless copies or clones of an organism or segment of DNA. Cloning produces a population that has an identical genetic makeup. Colony A clump of genetically identical bacterial cells growing on an agar plate. Because all the cells in a single colony are genetically identical, they are called clones. Culture Media The liquid and solid media referred to as LB (named after Luria and Bertani) broth and agar are made from an extract of yeast and an enzymatic digest of meat byproducts which provide a mixture of carbohydrates, amino acids, nucleotides, salts, and vitamins, all of which are nutrients for bacterial growth. Agar, which is from seaweed, polymerizes when heated and cooled to form a solid gel (similar to Jell-O gelatin), and functions to provide a solid support on which to culture the bacteria. Genetic Engineering The manipulation of an organism’s genetic material (DNA) by introducing or eliminating specific genes. 57 AP PE ND IX B Gene Regulation Gene expression in all organisms is carefully regulated to allow for differing conditions and to prevent wasteful overproduction of unneeded proteins. The genes involved in the transport and breakdown of food are good examples of highly regulated genes. For example, the simple sugar, arabinose, can be used as a source of energy and carbon by bacteria. The bacterial enzymes that are needed to break down or digest arabinose for food are only expressed in the absence of arabinose but are expressed when arabinose is present in the environment. In other words when arabinose is around, the genes for these digestive enzymes are turned on. When arabinose runs out these genes are turned back off. See Appendix D for a more detailed explanation of the role that arabinose plays in the regulation and expression of the Green Fluorescent Protein gene. Green Fluorescent Protein Green Fluorescent Protein (GFP) was originally isolated from the bioluminescent jellyfish, Aequorea victoria. The gene for GFP has recently been cloned. The unique three- dimensional conformation of GFP causes it to resonate when exposed to ultraviolet light and give off energy in the form of visible green light. When exposed to UV light, the electrons in GFP's chromophore are excited to a higher energy state. When they drop down to a lower energy state, they emit a longer wavelength of visible fluorescent green light at ~509 nm. Plasmid A circular DNA molecule, capable of self-replicating, carrying one or more genes for antibiotic resistance proteins and a cloned foreign gene such as GFP. It is an extra-chromosomal DNA molecule separate from the chromosomal DNA. Plasmids usually occur naturally in bacteria. pGLO Plasmid containing the Green Fluorescent Protein gene sequence and ampicillin resistance gene, which codes for beta-lactamase. Recombinant DNA The process of cutting and recombining DNA fragments Technology as a means to isolate genes or to alter their structure and function. Screening Process of identifying wanted bacteria from a bacterial library. Sterile Technique Minimizing the possibility of outside bacterial contamination during an experiment through observance of cleanliness and using careful laboratory techniques. Streaking Process of passing an inoculating loop with bacteria on it across an agar plate in quadrants with the intent of generating single colonies. Vector An autonomously replicating DNA molecule, such as a plasmid, into which foreign DNA fragments are inserted and then propagated in a host cell. 58 APPENDIX B The four bases in RNA are A, G, C, and U (uracil); the pairing rules are the same as for DNA except that A pairs with U. Although RNA can pair with complementary RNA or DNA, in cells RNA is usually single-stranded. The sugar in the RNA backbone is ribose, not deoxyribose. RNA molecules are generally short, compared to DNA molecules; this is because each RNA is itself a copy of a short segment from a DNA molecule. The process of copying segments of DNA into RNA is called transcription, and is performed by a protein called RNA polymerase. Proteins Proteins (more precisely, polypeptides) are also long, chain-like molecules but are more structurally diverse than either DNA or RNA. This is because the subunits of proteins, called amino acids, come in twenty different types. The exact sequence of amino acids along a polypeptide chain determines how that chain will fold into its three-dimensional structure. The precise three-dimensional features of this structure, in turn, determine its function. What a protein will do depends on the exact sequence of its amino acids. In most cases, a protein will perform a single function. Very diverse functions can be performed by proteins: Some proteins, called enzymes, act as catalysts in chemical reactions; some carry signals from one part of a cell to another—or, in the case of “hormones”, from one cell to another; some proteins (antibodies) have the task of fighting intruders; many become integral parts of the various physical structures inside cells; and still others (regulatory proteins) police various activities within cells so as to keep them within “legal” limits. Linear code, three-dimensional consequences DNA is the primary depot for information in living systems. As mentioned, this information is linear, i.e., encoded in the sequence of A, G, C, T building blocks along the DNA molecule. This linear code can be passed on to offspring—because DNA can be replicated in exact copies. Short segments of each DNA molecule are chosen for transcription at any given time. These segments are called genes. The enzyme, RNA polymerase, copies the entire segment, base by base, assembling an RNA molecule which contains a sequence of A, G, C and U exactly complementary to the DNA sequence of the transcribed gene. In addition to providing a master template for copying RNAs, DNA also contains sequence information which tells the RNA polymerase where to start transcribing a gene (promoter) and where to stop, how many copies it should make and when, and it can even embed certain information within the RNA sequence to determine the longevity and productivity of that RNA. There are three major classes of RNAs copied off DNA templates: messenger RNAs, or mRNAs, which relay the sequence information required for assembling proteins; transfer RNAs, or tRNAs, which work in the assembly line for proteins; and RNAs which perform structural functions. For example, ribosomal RNAs, or rRNAs, help build the scaffolding for ribosomes, the factories where proteins are assembled. mRNAs carry the sequence information for making proteins. Ribosomes read this sequence of nucleotides, by a process called “translation”, into a sequence of amino acids. How is this accomplished? There are only four kinds of nucleotides, but twenty kinds of amino acids. During translation, the ribosome reads 3 nucleotides at a time and assigns an amino acid to each successive triplet. Note: Triplets are often referred to as codons. Each amino acid is then attached to the end of the growing protein chain. There are 64 possible triplets, 61 AP PE ND IX C or codons. Thus, the linear information residing in DNA is used to assemble a linear sequence of amino acids in a protein. This sequence, in turn, will determine the way that protein will fold into a precise shape with characteristic chemical properties. In summary, the primary transfer of information within cells follows the order: DNA!RNA!PROTEIN!TRAIT Although the information itself is linear, the implications are three-dimensional. A fundamental assumption of recombinant DNA technology is that permanent and desirable changes in the functioning of living cells can be accomplished by changing the linear sequence of their DNA. Genes are discrete files of DNA information A gene is a segment within a DNA molecule singled out for copying into RNA. Directly or indirectly, this RNA will perform a function. It is convenient to think of a gene, therefore, as a unit of function. Many traits, such as bacterial resistance to an antibiotic, are governed by single genes. Most traits—such as the color of a rose, or the shape of a nose—are governed by several genes acting in concert. Genes can vary in length: Some are only a few hundred base pairs long; some can be tens of thousands of base pairs long. A DNA molecule may carry from a handful to thousands of genes. A cell, in turn, may contain one or several DNA molecules (chromosomes). Thus the number of genes in a cell can vary greatly. E. coli, a bacterium, contains one DNA molecule with about 5,000 genes on it. A human cell contains 46 DNA molecules carrying a total of about 100,000 genes. All genes in a given cell are not copied into RNA (i.e. “expressed”) at the same time or at the same rate. Thus, when speaking of gene function, one refers to its expression level. This rate can be controlled by the cell, according to predetermined rules which are themselves written into the DNA. An example: The cells in our bodies (all 100 trillion of them) each contain identical DNA molecules. Yet liver cells, for example, express only those genes required for liver function, whereas skin cells express a quite different subset of genes. DNA can be cut into pieces with restriction enzymes Restriction enzymes are proteins made by bacteria as a defense against foreign, invading DNA (for example, viral DNA). Each restriction enzyme recognizes a unique sequence of typically 4–6 base pairs, and will cut any DNA whenever that sequence occurs. For example, the restriction enzyme BamH I recognizes the sequence (5'..GGATCC..3') and cuts the DNA strand between the two G nucleotides in that sequence. Restriction enzymes will cut DNA from any source, provided the recognition sequence is present. It does not matter if the DNA is of bacterial, plant or human origin. 62 APPENDIX C Pieces of DNA can be joined by DNA ligase DNA ligase is an enzyme that glues pieces of DNA together, provided the ends are compatible. Thus, a piece of human or frog or tomato DNA cut with BamH I can be easily joined to a piece of bacterial DNA also cut with BamH I. This allows the creation of recombinant DNAs, or hybrids, created by joining pieces of DNA from two different sources. Genes can be cut out of human DNA or plant DNA, and placed inside bacteria. For example, the human gene for the hormone insulin can be put into bacteria. Under the right conditions, these bacteria can make authentic human insulin. Plasmids are small circular pieces of DNA Plasmids are small circular DNAs found inside some bacterial cells. They replicate their own DNA by borrowing the cells’ polymerases. Thus they can persist indefinitely inside cells without doing very much work of their own. Because of their small size, plasmid DNAs are easy to extract and purify from bacterial cells. When cut with a restriction enzyme, they can be joined to foreign DNAs—from any source—which have been cut with the same enzyme. The resulting hybrid DNAs can be reintroduced into bacterial cells by a procedure called transformation. Now the hybrid plasmids can perpetuate themselves in the bacteria just as before except that the foreign DNA which was joined to it is also being perpetuated. The foreign DNA gets a free ride, so to speak. Every hybrid plasmid now contains a perfect copy of the piece of foreign DNA originally joined to it. We say that foreign piece of DNA has been cloned; the plasmid which carried the foreign DNA is called a cloning vehicle or vector. In addition to their usefulness for cloning foreign genes, plasmids sometimes carry genes of their own. Bacteria die when exposed to antibiotics. However, antibiotic-resistance genes allow bacteria to grow in the presence of an antibiotic such as ampicillin. Such genes are often found on plasmids. When foreign DNA is inserted into such plasmids, and the hybrids introduced into bacterial cells by transformation, it is easy to select those bacteria that have received the plasmid—because they have acquired the ability to grow in the presence of the antibiotic, whereas all other bacterial cells are killed. DNA libraries When DNA is extracted from a given cell type, it can be cut into pieces and the pieces can be cloned en masse into a population of plasmids. This process produces a population of hybrid (recombinant) DNAs. After introducing these hybrids back into cells, each transformed cell will have received and propagated one unique hybrid. Every hybrid will contain the same vector DNA but a different insert DNA. If there are 1,000 different DNA molecules in the original mixture, 1,000 different hybrids will be formed; 1,000 different transformant cells will be recovered, each carrying one of the original 1,000 pieces of genetic information. Such a collection is called a DNA library. If the original extract came from human cells, the library is a human library. Individual DNAs of interest can be fished out of such a library by screening the library with an appropriate probe. 63 AP PE ND IX C Appendix E Photodocumentation of pGLO Plates Using Vernier’s BlueView Transilluminator 1. Start Logger Pro® and choose New from the File menu. 2. Prepare the BlueView Transilluminator. a. Transfer the +pGLO LB/amp plate to the central portion of the blue platform of the BlueView Transilluminator. b. Connect the BlueView Transilluminator to AC power and turn it on. 3. Positioning the ProScope HR™. a. Connect the 1–10X lens to the ProScope. b. Connect the ProScope to the USB port. c. Mount the ProScope to the stand and position the stand next to the transilluminator. d. Level the ProScope so that its lens is parallel to the surface of the transilluminator. 4. Prepare Logger Pro for use. a. Choose Add Page ! Blank Page ! OK from the Page menu. b. Choose Text from the Insert menus and enter a title to describe the test, for example “+ pGLO, Colonies LB/amp/ara.” c. Choose Video Capture ! Take Photo from the Insert menu. d. Orient and focus the ProScope HR so that colonies are sharply focused. 5. Place the Imaging Hood over the ProScope and the transilluminator. Reach through the flap of the hood to make final adjustments for best position, focus, and resolution. 6. When you are satisfied with the image, click on Take Photo and choose Auto Arrange from the Page menu. 7. The screen should now resemble Figure 1. 8. Add a new page and title for the next plate and take its photo. 9. Repeat for each subsequent plate. 10. Colony count data and plate information can be added to page 1 data table (optional). 66 APPENDIX E Figure 1 Appendix F References 1. Hanahan, Douglas, Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol., 166, 557 (1983). 2. Hanahan, Douglas, Techniques for transformation of E. coli. In DNA Cloning: A Practical Approach (Ed. D. M. Glover), vol. 1. IRL Press, Oxford (1987). 3. Schleif, Robert,Two positively regulated systems, ara and mal, In Escherichia coli and Salmonella, Cellular and Molecular Biology, Neidhardt. ASM Press, Washington, D.C. (1996). Parafilm is a trademark of American National Can Co. Jell-O is a trademark of Kraft Foods, Inc. Logger Pro is a trademark of Vernier Software & Technology. Pro Scope HR is a trademark of Bodelin Technologies. 67 AP PE ND IX F