1431489 REV 001 Neo/SCI Lab 8 Bacterial Transformation BACKGROUND INFORMATION What Is Biotechnology? Before you start doing biotechnology laboratory exercises, it is important to know exactly what biotechnology is and why it is useful. The first part of the word bio means living. The second part of the word technology means the practical application of knowledge. Thus biotechnology can be defined as the practical application of knowledge acquired from the study of living things. Many of these applications are not new to us. People have been making food, medicines, and other products using the unique properties of living organisms for centuries. Ancient Egyptians knew how to make wine and beer by fermenting grapes and grains. For centuries, humans have taken advantage of the characteristics of living organisms to make cheese and bread. We have known that it is possible to create corn that grows larger ears and cows that produce more milk by selectively breeding species with particular combinations of traits What s new about biotechnology is our understanding. Ancient Egyptians and early farmers didn t really know why and how fermentation or selective breeding worked. They learned by trial and error. Today, scientists understand what happens at each step of the complex biological and chemical process. These processes are carried out according to instructions provided by each cell s deoxyribonucleic acid (DNA). Once scientists understand the genetic code that regulates cell activity, they can look for ways to alter and manipulate cellular functions to manufacture specific products in a predictable and controllable way. Double Helix DNA Bacterial Transformation The scientific revolution in biology which started in 1973 by biochemists Stanley Cohen and Herbert Doyer, yielded techniques that enabled biologists to produce genetically engineered organisms. The two biochemists isolated a gene from the toad, Xenopus laevis, and transferred it to the bacterium Escherichia coli. The process of isolating genes from one organism, building a recombinant DNA molecule and inserting it into another organism is a field known as genetic engineering. The benefits of genetic engineering have been evident in agriculture, medicine, environment and many other areas. For example, most people diagnosed as having diabetes need to take a hormone called insulin. The old way of making this drug was to extract it from animal pancreatic glands followed by purification. Today the human gene for making insulin can be spliced into a plasmid which is used as a vehicle to transport it into bacteria cells which act as the host. The bacterial cells then express the insulin gene as its own and produce insulin. To insert a gene into a plasmid, restriction enzymes derived from bacteria are first used to cut apart the gene of interest. These enzymes recognize specific nucleotide sequences and cut the DNA at these sites into fragments with short sticky ends. Complementary sticky ends from different organisms can then be joined together to form a recombinant DNA molecule. 1
Guide Name... Bacterial cells contain a single chromosome, but in order to ensure survival, more information is usually needed than what is provided by the chromosome. This additional information is encoded in plasmid DNA. Plasmids are mini-chromosome, small circular DNA molecules found mostly in bacteria but also in select yeast strains. They contain genes that are useful Plasmid Human Cell Bacterial Cell Gene Sticky ends Bacterial Transformation Recombinant DNA DNA insertion Gene to the host, as well as genes that encode for antibiotic resistance. Such genes produce large amounts of enzymes that chemically neutralize the antibiotics. At any time, a spontaneous mutation can occur at a plasmid in the bacterial cell, which could give rise to an antibiotic resistant gene. During conjugation or bacterial mating, the plasmids replicate and are transferred between bacteria. This conjugation, or transfer of plasmids between bacteria, makes bacteria antibiotic resistant and thus more difficult for doctors to treat an infection. When microbes began resisting penicillin, scientists developed new antibiotics to fight them off. However, today scientists fear that they have reached near the end of new antibiotic development. The latest developed antibiotic is vancomycin, known as the drug of last resort. Some hospital staph (Staphylococcus aureus) infections are resistant to all known antibiotics, except vancomycin. However, vancomycin resistance has turned up in another commonly hospital found bacteria, Enterococcus. It is only a matter of time until Vancomycin-resistant staph infections will appear, because bacteria exchange resistant genes quite rapidly. Staphylococcus may pick up vancomycin resistant genes from Enterococci, which are found in the normal gut. LAB OVERIEW This guided and open investigation provides an introduction to some of the techniques used in the field of genetic engineering. In the model experiment you will perform a procedure that will transform a special strain of E. coli bacteria using a plasmid vector to express new genetic information that will confer ampicillin antibiotic resistance to the E. coli. The plasmid that will be used to transform the bacteria in this investigation is puc18 and occurs naturally in E. coli. It contains an ampicillin resistant gene that confers antibiotic resistance to bacterial cells. Transformed bacteria cells will grow in the presence of the antibiotic while bacteria cells that did not take up the plasmid, will not grow. Your teacher will assist you in using mathematical routines to determine the efficiency of the transformation process. Under your teacher s supervision you will then have the opportunity to design and conduct individual experiments to modify the transformation process by manipulating environmental conditions, or induce mutations with observable phenotypes. Amp Lac Z 2,686 BP Amp R = Ampicillin Resistance gene Lac Z = Gene for Galactosidase Figure 1: Plasmid puc18 A plasmid is a circular dsdna molecule. This plasmid has been genetically engineered to include a gene for antibiotic resistance to ampicillin (ampr) and a gene (and its promoter) for the enzyme beta-galactosidase (laz). 2
Guide Name... CORE CONCEPTS Does it matter what type of bacteria takes up a Big Idea 3: Living systems store, retrieve, transmit, and plasmid? respond to information essential to life processes. Can a bacterial cell take up more than one type of Genetic information provides for continuity of life, plasmid at the same time? and, in most cases, this information is passed from How could you determine the maximum expression of parent to offspring via DNA. a transformed plasmid? When the DNA of a cell changes, the RNAs and Do plasmids just confer antibiotic resistance or is there proteins they produce often change, which in turn another survival advantage? changes how that cell functions. Can bacteria express proteins from different organisms? DNA inside a cell can change several ways. It can be mutated, either spontaneously or after the DNA MODEL EXPERIMENT replication machinery makes an error. Under your teacher s instruction, you will perform a Biotechnologists may cause an intentional mutation bacterial transformation using E. coli strain JM101 with the in a cell s own DNA. DNA plasmid puc18. The student guide contains step-bystep instructions on how to prepare competent bacterial One common technology, bacterial plasmidbased genetic transformation, enables students cells which will take up the plasmid. Using a selective to manipulate genetic information in a laboratory media plate you will identify transformed colonies and setting to understand more fully how DNA operates. calculate the efficiency of the procedure. It is essential that you have a good understanding of the LEARNING OBJECTIVES techniques involved in manipulation of the bacteria as To demonstrate the universality of DNA and its well as the biochemical and cellular processes involved expression in the expression of the β-lactamase gene of the plasmid To explore the concept of phenotype expression which confers the antibiotic resistance necessary for the To explore how genetic information can be bacteria to survive in the presence of ampicillin. Given transferred from one organism to another this knowledge, you should be able to expand on this To investigate the concept of horizontal gene transfer procedure to investigate other variables in the process. and the subsequent increase in genetic variation Independent Inquiry Pathways To explore the relationship between gene expression After completing the model experiment, you will be given and certain environmental factors suggested paths to take for your Independent Inquiry To work collaboratively to define investigation Investigation. Your teacher will direct you to the options problems that you are able to choose from or may direct you to complete a particular activity. EXPERIMENTAL DESIGN CONSIDERATIONS To help you understand the material that you are Scientific inquiry will help you and your group members expected to apply in this activity, be sure you can develop skills in communication, teamwork, critical thinking, understand and discuss the following concepts before and commitment to lifelong learning. This investigation you start this lab activity. can help foster these skills. An important part of becoming Do all plasmids contain the same information? a scientist is to learn to keep clear, concise, and accurate laboratory notes. At the conclusion of the independent Are all plasmids the same size? investigations, your teacher will ask you to create miniposters that showcase the results of your Part A or Part B Is there a difference between genetically engineered and naturally occurring plasmid DNA? experiments or provide a formal lab report. An organized Do all plasmids transform bacteria with the same lab notebook should demonstrate originality and reflection efficiency? while serving as a record of your work. 3
KEEPING A LABORATORY NOTEBOOK A lab notebook allows you to organize your work for use in a later, formal report. It should contain: Work group members Primary question (stated problem) for investigation Background observations and contextual information Hypothesis and rationale for the investigation Notes on Procedure / Experimental Design strategies for testing hypothesis, using appropriate controls and variables Materials required Safety issues (or specific cautions) Procedure in sufficient detail so that another student group could replicate team results Results, including graphs, tables, drawings or diagrams, and statistical analysis Conclusion and discussion Was the hypothesis supported? What additional questions remain for further investigation? Citations for sources found through library or web research SAFETY & DISPOSAL Always follow proper lab safety and aseptic technique protocols. Wear protective gloves, goggles, and a lab apron when working with bacteria. Once your petri plates have been inoculated with bacteria, seal them with clear tape and do not open them. When working with bacteria, do not touch your face or mouth with your hands. Keep your work area clean. At the end of the investigation, collect all petri plates and all contaminant items such pipets, microfuge tubes, etc. Prior to disposal, your teacher may instruct you to sterilize all contaminated materials by immersing them in a 5% bleach solution for about 1 hour. Your work area should be disinfected by wiping it with either a bleach solution or 95% ethanol. Dispose of all materials as instructed by your teacher. Wash your hands immediately after handling bacteria and before leaving the laboratory. GETTING READY Prior to beginning the model experiment, read through, or view the PowerPoint presentation Lab Skills to thoroughly understand what is involved in Experimental Design, Defining a Problem, Keeping a Laboratory Notebook, and Identifying a Variable. It is essential that you are familiar with the techniques involved in manipulating DNA, bacteria and to have a reasonable understanding of what is involved in the model experiment. You should be very clear on what to do and what not to do when following a procedure which requires sterile technique so as not to introduce a contaminating microorganism. The tutorial on Bacterial Transformation presents the general procedure used to transform bacteria: http://www.youtube.com/results?search_ query=bacterial+transformation After watching the tutorial, your teacher may have you practice the following activities as a dry run prior to beginning the lab: Using an inoculating loop to streak a plate. This does not have to be done with the bacteria, but you should be skilled and careful enough not to gouge the agar with the loop. Using a pipette to transfer and deliver specific volumes of liquid, making certain that you understand the gradations and volume markings on the exterior surface. THE INVESTIGATION Bacterial Transformation Ampicillin Resistance THINK ABOUT IT... Answer these questions in your lab notebook before completing the Investigation. Explain the process of transformation. What are plasmids? How are they transferred? What is genetic engineering? How is it used? List some examples of genes found in genetically engineered plasmids. Can bacteria express proteins from different organisms? Can a bacterial cell take up more than one type of plasmid? 4
(Per Student) Gloves Apron Goggles WHAT YOU NEED (Per Group) Marker 2 2-mL Sterile microfuge tubes Microfuge tube rack 2 Sterile 1 ml micropipets 500μL 0.05 M CaCl₂ (on ice) 1 Sterile inoculating loop 10μL puc18 solution 2 Luria broth agar plates 2 Luria broth agar with ampicillin plates 500μL Luria broth 1 Spreading rod 1 Digital or fixed volume micropipet (with at least 5 tips) Clear tape (Per Class) E. coli culture plate Ice bath 42 C Water bath Rubbing alcohol Flame source Incubator set at 37 C STEP 1 Obtain two poured Luria broth plates. Label one of the plates LB+, and the other plate LB- STEP 2 Obtain two poured Luria broth with ampicillin plates. Label one of the plates LB/Amp+, and the other plate LB/Amp-. STEP 3 Obtain two sterile 2-mL microfuge tubes. Label one of the tubes +, and the other tube -. STEP 4 Using one sterile, plastic 1-mL micropipet, add 250 μl of ice cold 0.05 M CaCl 2 to each of the sterile microfuge tubes. STEP 5 Using a sterile, plastic inoculating loop, add an E. coli colony of approximately 3 mm in diameter to each of the two-2 ml microfuge tubes. Try not to gouge the agar that the E. coli are growing on as no agar should be introduced to the tubes. Tap the inoculating loop firmly against the side of each tube to ensure that a colony is introduced to each tube. Discard the inoculating loop as directed by your teacher. STEP 6 Use the micropipet from Step 4 to suspend the cells in the CaCl 2 in the tubes. Do this by alternately drawing the solution from the tube into the micropipet and emptying it several times. Discard the micropipet as directed by your teacher. STEP 7 WHAT TO DO Using a digital or fixed volume micropipet, introduce the antibiotic-resistance plasmid by adding 10 μl of puc18 solution into the + tube, and mix the contents by tapping on the tube. 5
STEP 8 Place both microfuge tubes directly in the ice container for a total of 15 minutes. STEP 9 Heat shock the bacterial cells by placing both tubes in a water bath at 42 C for a total of 90 seconds. STEP 10 Return both tubes immediately to the ice container, and allow them to remain there for 2 minutes. STEP 11 Using one sterile, plastic 1-mL micropipet, add 250 μl of Luria broth to each of the microfuge tubes and tap on each tube to mix the contents. Place the tubes in the microfuge tube rack at room temperature. Discard the micropipet as directed by your teacher. STEP 12 Using a digital or fixed volume micropipet, draw 100 μl of + cells and dispense them onto the LB+ plate. Place 100 μl of + cells on the LB/Amp+ plate also. STEP 13 Sterilize the spreading rod by dipping it in alcohol and flaming it. Touch the rod to the surface of the agar (an area without cells) to cool it, and then spread the + cells across each of the two + plates. Cover the plates immediately and place them aside to set for a few minutes. Resterilize the spreading rod with alcohol and flame. STEP 14 Using a clean tip on the digital or fixed volume micropipet, draw 100 μl of - cells and dispense them onto the LB- plate. Place 100 μl of - cells on the LB/Amp- plate also. STEP 15 Resterilize the spreading rod and use it to spread the - cells across each of the two - plates. Cover the plates immediately and place them aside to set for a few minutes. Resterilize the spreading rod as you did previously. STEP 16 All plates should be taped with clear tape at a few places around the rim, and incubated inverted in a 37 C incubator overnight. Note: The petri plates should not be completely sealed as this would create anaerobic growth conditions DATA SUMMARY AND ANALYSIS Observe each of the incubated plates. Do NOT open them; colonies are visible through the bottom of the plates. Count the total number of colonies present on each plate and enter those values in Data Table 1. If the colonies are too numerous to count (if the colonies have grown into each other), this is called a lawn. Record this in Data Table 1. LB+ LB- LB/Amp+ LBAmp- Plate DATA TABLE 1 Number of Colonies The total number of antibiotic-resistant bacterial colonies visible on each plate can be used to calculate the transformation efficiency, or the number of antibioticresistant colonies/μg puc18. Perform the following calculations to determine the transformation efficiency. In this procedure, you used a 0.005 μg/μl concentration. You used a total volume of 10 μl puc18. Calculate the total mass of the puc18 used: Total mass of puc18 (mass = volume x concentration): Total volume of cell suspension: Fraction of the total cell suspension spread on plate (μl spread/total volume): Mass of puc18 in the cell suspension (total mass x fraction spread on plate): The transformation efficiency is the number of colonies/μg plasmid (# colonies observed/mass of puc18 in suspension): (use scientific notation) 6
QUESTIONS 1) Three of the plates were controls. Which three plates were the controls? For each, describe how it was a control. 2) Make a pairwise comparison of the following plates: LB+ and LB-; LB+ and LB/Amp+; LB- and LB/ Amp-; LB/Amp+ and LB/Amp-. Describe the growth on each pair of plates and discuss why the growth appeared as it did.... 3) Explain the reason for heat shocking the bacteria. 4) How do you determine whether a bacterium has been transformed? 5) How can you differentiate a satellite colony from a transformed colony? 6) What is the reason for culturing the bacteria at 37 C? 7) List the steps in the procedure that require sterile technique or materials. 8) Explain why sterile technique or materials are necessary in those steps. 7
INDEPENDENT INVESTIGATION INQUIRIES Now that you have completed the model investigation you will be asked to design and carry out an Independent Inquiry Investigation. It should be an extension of the experiment that you just completed. What are some variables that you can alter? What can you change to modify or improve the experiment? Brainstorm some ideas with your classmates. Make notes and observations in your Notebook as you go along. Below are listed some suggested problems for you to investigate. Your teacher will give you direction on which of the options you may use. Part A: 1. What is the effect of altering the length of the heat shock or changing the ph or concentration of the CaCl 2 solution? 2. Determine the upper limit of antibiotic resistance by increasing the concentration of ampicillin in a series of LB plates. 3. Does having the puc18 plasmid give the transformed bacteria an advantage over the untransformed bacteria other than antibiotic resistance? Part B: 1. Compare transformation efficiency using pbr322, a larger, ampicillin resistant plasmid. 2. Compare transformation efficiency using puc57, a smaller tetracycline resistant plasmid. 3. Transform the bacteria with puc18 and puc57 to determine if the bacteria can take up more than one plasmid at a time. 4. Perform a novel transformation experiment to show that the bacteria produce a protein which is not part of their original genetic complement. Once you have identified the problem you will investigate you need to design an experiment. Using the information you have learned in class and in the PowerPoint presentation, write out a procedure for your experiment. Be sure to have your teacher check your work before you begin your experiment. Record your procedure in your lab notebook and notes and observations as you go along. You will use this information to either create a mini-poster or a formal lab report. Detailed notes will be invaluable for this part of the process. EVALUATING Your teacher will instruct you on what type of assessment you will be completing at the end of your Independent Investigation Inquiry. GOING FURTHER The background to this investigation asks you to think about several applications of genetic transformation, including genetically modified food and possible ethical, social, or medical issues raised by the manipulation of DNA by biotechnology. Discuss why these issues are issues. What questions are posed by genetic engineering? Respond to the quote from Michael Crichton s novel and film Jurassic Park: Just because science can do something doesn t mean that it should. LEARN MORE ABOUT IT Small Scale Isolation and Agarose Gel Purification of Plasmid DNA http://www.youtube.com/watch?v=v7w-disxpm8 Genetic Transformation of Plants http://www.scribd.com/doc/43766647/genetic- Transformation-in-Plants Investigate other means by which DNA is moved into an organism to alter its genetic makeup 8
Genetic Transformation of Mammalian Cells http://www.molecularstation.com/transfection/ Investigate the use of (cationic) polymers, liposomes and nanoparticles methods of introducing foreign DNA into a eukaryotic cell. Transgenic Animals-Key to Human Disease Research http://www.ctcase.org/bulletin/11_3/transgenic.html Recombinant Techniques for Producing Transgenic Animals http://users.rcn.com/jkimball.ma.ultranet/biologypages/t/ TransgenicAnimals.html Transgenic animals carry foreign gene(s) that has been strategically inserted into their chromosomal DNA. In addition to the gene itself, the DNA usually includes other sequences that enable it to be incorporated into the DNA of the host animal s cells and to be expressed correctly by those cells. Biotechnology Chromosome Conjugation Deoxyribonucleic acid GLOSSARY Use of living systems and organisms to develop or make useful products in agriculture as well as food and medicine production A piece of genetic material that contains DNA and associated proteins A process where two bacteria cells temporarily join together and transfer a plasmid from the donor cell to the recipient cell A double stranded nucleic acid that contains the genetic information for cell growth, division and function DNA Genetic engineering Microbes Plasmid Recombinant DNA Restriction enzymes Selective breeding Abbreviation for deoxyribonucleic acid Manipulation of an organisms genome using modern DNA technology A microscopic organism such as a bacterium, fungus, protozoan or virus A circular piece of DNA that is capable of replicating independently from the chromosomal DNA DNA formed from two or more different sources that have been cut and joined together An enzyme that causes the cutting of DNA at certain specific places called restriction sites Intentional breeding of organisms with desirable traits in an attempt to produce offspring with similar desirable characteristics 9