Genetic transformation LAB REPORT

HELP IN DOING A LAB REPORT ABOUT GENETIC TRANSFORMATION .MOST INFORMATIONS ARE PROVIDED IN THE ATTACHMENT Laboratory 5: Genetic transformation & green fluorescent protein Objectives: In this lab you will alter the genetic program of the bacterium Escherichia coli by adding a gene from the jellyfish Aequorea victoria. You’ll be exploring the relationship between DNA sequence and gene expression, so you’ll need to understand what a plasmid is, how the genes are arranged on the plasmids, and how the genes can be turned on and off. You’ll also be using the green fluorescent protein (GFP) as an easy indicator of gene expression. After this lab, you should be able to give a molecular explanation for how specific genetic modification can change the phenotype of a living organism. Genetic transformation and plasmids When DNA was identified as the carrier of genetic information, it was found that an organism could be induced to have new characteristics, or phenotypes, by simply adding new DNA to the organism. The term used for this alteration was ‘transformation’, and this term is still in use to describe experiments in which new DNA is added to a cell. Initial experiments in transformation were performed by Frederick Griffith and Oswald Avery in the 1920s through the 1940s, and involved the natural transformation of non-pathogenic bacteria with virulence genes from pathogenic bacterial donors. The first deliberately created recombinant DNA was reported by Paul Berg and colleagues in 1972, and involved a virus (SV40) that was engineered to contain bacterial genes. Following this experiment, further experiments in genetic engineering were halted until regulations could be debated and agreed upon by the scientific community and government officials. The Asilomar Conference on Recombinant DNA took place in 1975, and led to the guidelines for recombinant DNA research that are currently in effect for government-funded research in the United States (see http://oba.od.nih.gov/rdna/rdna.html for details). Organisms that contain foreign genes are described as transgenic, or genetically modified. Transgenic organisms are now commonplace in biomedical research and biotechnology, and have also entered the marketplace. For example, many crops containing genetic modifications are grown throughout the world. In addition, ‘Glo fish’, zebra fish that express different colors of fluorescent proteins, are available for purchase at local pet stores. In medicine, human genes can be altered through the process of gene therapy. However, the recipients of gene therapy are not formally referred to as transgenic, since their germ lines (the cells that give rise to eggs or sperm) are not genetically altered, because of important ethical considerations. To move foreign DNA into an organism, a vector is often used. Vectors are pieces of DNA that contain the foreign gene, as well as sequences that maintain replication in the host organism, and allow the foreign gene to be expressed properly. Examples of vectors include non-pathogenic viruses, as well as small circular pieces of DNA called plasmids. Plasmids are naturally occurring DNA elements that contain sequences allowing replication in a bacterial cell. 41 In this lab you’ll use two different plasmids, shown in Figure 1. The plasmid pGFP was constructed using standard molecular biology techniques that allow cutting and pasting of DNA fragments. The pGFP plasmid is an example of a recombinant vector, since it contains foreign DNA: in this case, a gene from a jellyfish that produces a fluorescent protein. Figure 1. Two plasmids for Escherichia coli. pUC19 is a very small plasmid with the gene for beta lactamase, giving resistance to the antibiotic ampicillin. pGFP also contains the beta lactamase gene, and has the green fluorescent protein gene under control of the pBAD promoter. The AraC DNA binding protein controls the pBAD promoter and regulates expression of green fluorescent protein. Once a recombinant plasmid vector is prepared, it needs to be added to the target organism. In many cases, it is necessary to treat the target organism in some way to make it competent, which means it is able to take up DNA from its environment. Bacterial cells can be made competent either by chemical treatment or through an electrical shock. In this lab, E. coli will be made competent for DNA uptake by treatment with magnesium ions, which are thought to damage the cell surface and allow DNA transient access to the cytoplasm. Transformation is an inefficient process. In a typical bacterial transformation experiment, less than 1% of the cells take up the foreign DNA, and it can be difficult to find those cells and separate them from the the cells that did not receive the DNA. To solve this problem, most vectors contain a selectable marker, which is a gene that the cell needs to survive. In this lab, the selectable marker is the gene for beta lactamase, an enzyme that breaks down the antibiotic ampicillin. If an E. coli cell contains the plasmid, it makes beta lactamase and breaks down the ampicillin, allowing cell survival. Cells that do not have the plasmid are not so lucky, and are killed by the ampicillin. In short, only those cells that receive the plasmid will survive when grown on media containing ampicillin. Expression of a transforming gene When a foreign gene is introduced into an organism, it will not be expressed unless specific control DNA sequences are present. The pGFP plasmid expresses the green fluorescent protein gene, because the pBAD promoter causes RNA polymerase to transcribe the gene (DNA ??RNA), and a ribosome binding site (not shown) causes translation of the GFP mRNA (RNA?protein). 42 It’s important to understand that the pBAD promoter is itself controlled by the transcriptional activator AraC (see Figure 2). AraC is a protein that can interact with DNA at the O2 site as well as at the two I sites, I1 and I2. The interaction mode of AraC is determined by the presence of the 5 carbon sugar arabinose. When no arabinose is present, two AraC proteins interacts with O2 and I1, and transcription does not occur. When arabinose is present, AraC partially re-folds, and now the two AraC proteins interact with I1 and I2. In this second configuration, RNA polymerase (RNAP) can make RNA, and the gene (off to the right, and not shown) gets expressed. AraC Figure 2. The pBAD promoter and its control by AraC and arabinose. AraC is a DNA binding protein whose folding is influenced by the sugar arabinose. The AraC I1/I2 binding mode allows RNA polymerase to transcribe the gene (in the rightward direction). Figure reproduced from Schleif (2010) FEMS Micro. Rev. 34, p. 779. Green fluorescent protein Research in cell biology has been greatly aided by the introduction of the green fluorescent protein (GFP), which provides an easily visualized marker for gene expression. The GFP gene was initially isolated from the jellyfish Aequorea victoria in 1992. The structure of GFP includes a unique serine/tyrosine/glycine triad that combine to form a fluorescent chromophore. The intensity and color of fluorescence produced by the chromophore is influenced by the rest of the GFP protein sequence, and the cloned version of GFP was altered by mutation to make the protein fluoresce brighter, and produce new colors. By the year 2000, GFP and related proteins became a very common tool in cell biology and microscopy. Cellular proteins can be easily tagged with GFP, which allows these proteins to be visualized by fluorescence microscopy. Many striking images of cellular structures have been enabled by these protein-GFP fusions. For the purposes of this lab, GFP provides an easily scored indicator of transgene expression following transformation. No transcription Transcription 43 KEY CONCEPT?Transformation of a living system with new genes means that the genotype (the genes present) and often the phenotype (the outwardly measurable characteristics) of the organism are changed. The phenotype of the transformed organism can tell you how the transgene is expressed. Your challenge: The bacterium Escherichia coli has served as a model organism for working out the basics of genetic transformation and gene expression over the past several decades. E. coli can be easily transformed with foreign DNA using a number of methods. You will chemically treat a culture of E. coli cells to make them competent (ready to take up foreign DNA). The chemically treated cells will then be mixed with various kinds of DNA, and you will make predictions of phenotypes based on the forms of DNA you transform with, and the kind of growth medium the transformed cells are incubated on. Prior to coming to lab, propose a hypothesis about whether circular or linear DNA fragments transform cells better. Also, propose a second hypothesis about the relationship between the expression of the green fluorescent protein and the presence of the sugar arabinose. Work in groups of two to compare hypotheses, and to complete the experiments suggested by these hypotheses. Materials provided: I. A culture of Escherichia coli that is sensitive to the antibiotic ampicillin, and is not fluorescent. Note: this strain of E. coli is non-pathogenic, meaning it does not cause disease. However, federal guidelines stipulate that the bacterial waste produced by this experiment should be disposed of as biohazardous, so dispose of all used materials in the red containers in the lab. Be sure to wash hands with soap after finishing in the lab, and wipe down bench surfaces with antibacterial cleaner after you are finished in the lab. ? II. Agar growth medium that can support bacterial growth, with each of the following additives (1 of each): a. The antibiotic ampicillin at 100 micrograms/milliliter (but no arabinose). This plate is marked with 1 green line on the side. ? b. The sugar arabinose at 0.5% (w/v) concentration (but no ampicillin). This plate is marked with 1 magenta line on the side. ? c. Ampicillin at 100 ug/ml, and arabinose at 0.5% (w/v) concentration. This plate is marked with 3 blue lines on the side. ? d. Ampicillin at 100 ug/ml, and arabinose at 0.05 % (w/v) concentration. This plate is marked with 3 orange lines on the side. ? III. 2X TSS solution, which contains 20% (w/v) polyethylene glycol (MW 3350), 10% (v/v) dimethyl sulfoxide (DMSO), and 100 mM MgCl2, at pH 6.5. ? 44 IV. Transforming DNA a. pUC19, circular, 25 nanograms ? b. pUC19, linear, 25 nanograms ? c. pGFP, circular, 25 nanograms ? V. A lamp that produces long wave ultraviolet (UV) light, in approximately the 365 nanometer range. ? Use the following information as you plan and then conduct your experiment: . 1) You may want to test several different transformation conditions and controls on each plate. If so, be sure to mark your plate into sectors to keep straight which transformation is being streaked on each sector. Only mark the plates on the bottom surface (the side containing the medium), NOT on the lids (in case the lids get mixed up). ? . 2) When labeling a plate, be sure to indicate the kind of medium in the plate (Does it have the antibiotic? Does it have the sugar?), the transformation tested in each sector, and write your name, your TA’s name, and the date on each plate. ? . 3) To transform the cells, obtain a fresh 1.5 ml capped tube, and label the lid appropriately. Add the DNA you want to transform with to the tube, or no DNA if you are doing this control experiment. Next, add 50 microliters of E. coli cells to the tube. Finally, add 50 microliters of ice-cold, 2X TSS to the tube. Mix the TSS and the cells and the DNA (if present) by pipetting up and down a few times. Leave the mixture in the tube and on ice for 10 minutes (a little longer is also OK). ? . 4) Totransfersomeofyourtransformationmixturetotheappropriateplate,and the appropriate sector on the plate, use a sterile, disposable loop. Dip the loop into the transformation mixture, and you’ll see a thin film of the mixture carried in the loop. Touch the loop to the surface of the medium in the plate, and then carefully swish the loop back and forth over the surface of the chosen sector until the entire sector is covered with trails from the loop. ? . 5) In your notes, make predictions for each sector. Will anything grow, and if so, why? If something does grow, will it show expression of the green fluorescent protein? How will you be able to tell? ? . 6) How will you know if the antibiotic is working as it should (ie. by killing bacterial cells)? ? . 7) How will you know if the plasmid gets transformed into cells? ? . 8) How will you know if the GFP gene is expressed in the transformed cells? ? 45 9) After you plate your transformations, your plates will be incubated at room temperature for a week. In your next lab session, retrieve your plates and examine the results. Check for fluorescence by using an ultraviolet light source. Record the number of colonies in each sector, and compare the results from all the different plates, and all of the different transformations. A few useful definitions?Genotype: The DNA sequences that an organism contains. Anything that changes the DNA represents a change in genotype. Phenotype: The outwardly measurable characteristics of an organism. Changes in genotype sometimes lead to a change in phenotype, but not always. Antibiotic: A small molecule that kills or halts the growth of bacteria.?Ampicillin: A specific antibiotic that is related to penicillin. Ampicillin kills bacteria by inhibiting synthesis of the peptidoglycan cell wall. Transformation: The introduction of foreign DNA into an organism. The new DNA changes the genotype, and often the phenotype of the organism. Competence: The ability of an organism to take up foreign DNA from the environment. Many bacteria are naturally competent, meaning that they take up DNA without any pre-treatment. E. coli requires chemical or electrical treatment to become competent for transformation by foreign DNA. Plasmid: Promoter: A small, circular piece of DNA naturally found in bacteria. Plasmids replicate independently of the cell’s chromosome. They typically contain genes that confer a selective advantage to the cell. A DNA sequence that allows a gene to be expressed. The promoter permits transcription of the gene (DNA ??RNA), and the RNA that is made is then translated (RNA?protein) by a ribosome. The protein folds, and a change in phenotype is often observed through the functioning of the protein. The pBAD promoter was identified in E. coli and is controlled by the action of the sugar arabinose. Activator of transcription: Promoters are usually subject to some kind of control by proteins that bind to DNA (DNA binding proteins). The AraC protein (produced by the araC gene) can activate (increase the function of) the pBAD promoter. Arabinose: A five carbon sugar that can be used as a food source by E. coli. Arabinose can trigger synthesis of genes require for arabinose utilization by affecting DNA binding by AraC. 46 Inducer: A small molecule that affects the DNA binding activity of a transcription factor. Arabinose is an inducer that affects AraC DNA binding activity. Questions . 1) (2 points) Why is the antibiotic ampicillin important for plasmid transformation? ?What’s the antibiotic doing, and what if it isn’t there following transformation? ? . 2) (2points)Whichphenotype(s)ofE.colidoespUC19affect?Whichphenotype(s) does pGFP affect? Which controls in your experiment allow you to be certain of these claims? ? . 3) (2 points) What was your hypothesis regarding the effect of DNA form on transformation? Is the form of the plasmid DNA important for transformation? How could you tell? ? . 4) (2points)Whatwasyourhypothesisregradingarabinose?Whateffectdoes arabinose have on cells that contain pUC19? What effect does it have on cells that contain pGFP? Does the concentration of arabinose have any effect on these phenotypes? ? . 5) (2 points) Many transgenic organisms have been created that contain the GFP gene. Based on your observations here, is expression of the green fluorescent protein harmful to cells? Which observations led you to your answer? ? Lab 6 hypothesis and pre-lab Questions (5 points) To receive full credit for this week’s lab write-up, prepare a hypothesis for the experimental outcome and answers to questions A through C for the next lab, Lab 6 (see page 56). 47 Lab 5 Draft Formal Lab Report The first draft should not be regarded as a rough draft, but as a complete report that represents your best effort. With a strong draft here, you’ll receive more useful feedback from your TA, and will have an easier time producing a polished final draft. The first draft will be graded as follows. Missing or deficient sections will result in point deductions. a. (2 points) Title and Introduction. ? b. (2 points) Hypothesis and predictions. ? c. (2 points) Experimental Procedure. ? d. (2 points) Data/Observations/Results. ? e. (2points)ConclusionsandReferences. ?Formal Lab Report, Draft (10 points) ? Scientific studies are reported to the public through the scientific literature, following a process of peer review and professional editing. A typical scientific report consists of a series of parts, including: 1) 2) 3) 4) 5) the Title of the study; an Introduction that describes the scientific question being studied, and how the study addresses it (this section often includes a Hypothesis); a description of the Experimental Procedures used to test the hypothesis; a report of the Results from the experiments;?a Conclusions/Discussion section that explains what the results mean relative to the hypothesis, and any new insights that arise from the study; and a list of References cited in the writing of sections 1) through 5). 6)?All scientific reports follow some version of this conventional format, helping ensure complete reporting by the scientist, and helping the reader find relevant information in the report quickly. Following the format detailed below, write a short, structured report to describe what you have done. Your report should be no more than 2 pages, single-spaced, 12 point type. Each section (except the title) should be given the headings indicated below, in boldface type. Be accurate and complete, but concise. Title: (0.5 pt)?Your lab report must have a title that is descriptive, clear, and concise. Introduction: (1.5 pts)?The Introduction section should be one short paragraph in length and include: the primary question being asked for the experiment, with appropriate background information already known about the question. Think of answering these questions in this section: “What is your question, why is it a question in biology (what do we lack an understanding of), and why is the question important?” Hypotheses: (2 pt) The hypothesis is generally included at the end of the Introduction section, but for this?48 report it should be separate. A hypothesis should be stated as a truism related to the question posed in the Introduction, a statement of how things work, and NOT as a prediction. Make the hypothesis present tense. Experimental procedure: (2 pts)?This section should include: Predictions that arise from your hypothesis, and how you tested those predictions through experiment in the lab. Include your experimental design and methods used to test your predictions. Make sure to describe your controls! Questions you should aim to answer in this section are: “What did you do? How did you do it?” Data/observations/results: (2 pts)?This section will contain a summary of your results, including graphs and tables. Be sure to include: An introductory text description of all data (State general results, and refer to tables and/or graphs for details) Try to answer the question “What did you see/record during the experiment?”, but without discussing what the data means. Be descriptive. Any table/figure/graph/drawing that you choose to include must contain captions. The tables and figures you include in your report should all be specifically described in the results section. Graphs must be a clear representation of your data, with properly labeled axes. An example of an acceptable graph with a caption is provided below. Hint: Captions indicate a figure number (to reference in the text of the results section), and a description (to help to fill in the blanks about what the graph is saying). The question being answered by a caption is “What data is represented in this graph?” Conclusions: (1 pts)?This section explains whether the results support or do not support your hypothesis. Here is where you explain what the data presented in the results section means. Identify if your hypothesis was supported or refuted. If the hypothesis was refuted, attempt to explain why this may be. If there were any procedural errors or potential weaknesses in the experimental design that may have influenced your data collection, mention them here. Try to answer the following questions in the section: “What does the data mean, is your hypothesis supported and why, and how do your results address the question posed in the background section?” You should also be able to pose a question to follow up what the results tell you. 49 References: 1 pt)?This section will contain any references that you have used in writing your report. You should include the lab manual in your list, as well as any other resource you found valuable in researching and writing the report. APA format for references is most appropriate, but MLA format is also acceptable. Additional Information Points will be subtracted for messy, unclear or poorly organized lab reports. Points will also be subtracted for grammar errors, so proofread your work! Reports longer than 2 pages will be graded on information in first 2 pages only. The draft report for lab 5 is due at the start of lab 7. Late lab reports will NOT be accepted.