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BIO208: GENETICS Bacterial Transformation with pGLO plasmid Cloning of GFP gene ASSIGNMENTS Read the terms and concepts portion of this laboratory Complete the pre-lab assignment due before the laboratory Record purpose, all steps, observations, and data in the laboratory notebook. Answer questions as indicated 2 Transformation of E. coli with pGLO Plasmid DNA With pGLO transformation, students transform bacteria with a gene that codes for a green fluorescent protein (GFP). The natural source for the GFP gene is the bioluminescent jellyfish, Aequorea victoria. The gene encodes for the GFP which allows the jellyfish to glow in the dark. The protein absorbs energy when exposed to ultraviolet light and gives off some of this energy in the form of visible green light. Genes can be moved into bacteria by the use of small circular pieces of DNA called plasmids. Plasmids may confer beneficial traits to a bacterium, such as resistance to ampicillin or other antibiotics. The pGLO plasmid encodes for resistance to ampicillin as well as a gene regulation system to control the expression of GFP. Expression of GFP by transformed bacteria will result in green glowing colonies. Those bacteria that do not take up the plasmid DNA will not produce GFP. pGLO plasmid vector bla orf GFP araC Encodes resistance to ampicillin for antibiotic selection Open reading frame allows plasmid to be copied by bacterial DNA replication enzymes Gene encoding green fluorescent protein Encodes a repressor to prevent transcription (and expression) of the GFP gene. The arabinose sugar binds the repressor to allow GFP expression. This allows the research to turn on and off GFP expression by addition or removal of arabinose from the bacterial growth medium. Transformation E. coli bacterium plasmids E. coli DNA Bacterial cell wall and cell membrane 3 Terms and Concepts Antibiotic selection The pGLO plasmid also contains the gene for ampicillin resistance. This gene encodes for beta-lactamase (bla) which is secreted by the bacteria harboring the plasmid. The beta-lactamase inactivates the ampicillin contained on the LB agar plates thereby allowing the bacteria to grow. Non-transformed cells (do not the plasmid) are killed by ampicillin. Ampicillin Provided as a 10 mg/ml stock in sterile water Ampicillin is a derivative of penicillin. This antibiotic kills bacteria by blocking the synthesis of a key cell membrane component. Ampicillin-sensitive cells are killed as they divide. Bacteria often carry small circular pieces of DNA called plasmids. The plasmid used in this lab contains an ampicillin resistance gene that encodes for beta-lactamase, a protein which inactivates ampicillin. Bacteria that carry the plasmid can be selected for by growing bacteria in the presence of ampicillin. Arabinose Arabinose is a sugar that bacteria can use as a source of carbon for cellular respiration. The enzymes needed to metabolize arabinose are only made in the presence of arabinose. The genes for the enzymes are part of the arabinose operon, a series of genes whose expression is coordinated and whose transcription is turned on by the presence of arabinose. When the arabinose is used up, the genes are no longer expressed. A gene of interest can be spliced onto the arabinose operon so that the expression of the gene is turned on in the presence of arabinose. Provided as a 200mg/ml stock in sterile water Beta-lactamase see antibiotic selection Cloning A population of cells produced from a single cell contains identical clones. The process of producing a clonal population is called “cloning”. One can produce large quantities of a specific DNA sequence or plasmid by cloning. Colony A group of identical bacterial cells (clones) growing on an agar plate. The colony contains millions of individual cells. Competent cells E. coli that have been treated with calcium chloride more readily accept plasmid DNA. Ca+2 ions are thought to neutralize the phospholipids of the cell membrane allowing the DNA to pass through the bacterial cell wall and enter the cell. E. coli The host organism in this experiment is E. coli strain K12. It is not pathogenic. However, Standard Microbiological Practices should be used. These bacteria grow best with aeration, provided by constant shaking. After 30 minutes of lag phase, E. coli enter a log phase in which doubling occurs every 20 minutes. When nutrients are depleted (cells at a concentration of 109/ml) cells enter a stationary phase. An overnight, stationary, culture of cells is used to prepare a mid log phase culture. LB at room temperature is inoculated with the overnight culture at a 1:100 dilution and allowed to incubate (with shaking at 37oC) for 1.75 hours. At this phase they exhibit an optical density of 0.4 at OD 550. Cells are then stored on ice for up to 24 hours to arrest in mid log phase. 100 ml midlog cells yield 10 ml competent cells. 200 ul cells are used for each transformation. 4 Gene Regulation Genes involved in the transport and breakdown of arabinose are highly regulated. The genes encoding the bacterial enzymes needed are only transcribed in the presence of arabinose. When the arabinose runs out, the genes are turned off. Green Fluorescent Protein GFP was originally isolated from the bioluminescent jellyfish, Aequorea victoria. The gene for GFP has been cloned and plasmid vectors containing the gene are in wide use. The 3D structure of GFP causes it to give off energy when exposed to UV light in the form of visible green light Heat Shock The heat shock increases the permeability of the bacterial cell membrane to exogenous DNA. The time of the heat shock is critical and is optimized for the tube type volume LB media Liquid and solid media are made from an extract of yeast and an enzymatic digest of meat byproducts which provides a mixture of carbohydrates, amino acids, nucleotides, salts, and vitamins – nutrients for bacterial growth. LB agar (made from seaweed) provides a support on which bacterial colonies can grow Plasmid A small circular DNA molecule capable of autonomous replication Many plasmids also contain antibiotic resistance genes and must contain an origin of replication. pGLO pGLO is a plasmid which contains the GFP gene and the ampicillin resistance gene which encodes for beta-lactamase Recovery The 10 minute incubation following the addition of LB broth allows the cells to grow and express the ampicillin resistance protein, beta-lactamase. Sterile technique It is important not to introduce contaminating bacteria into an experiment. Because contaminating bacteria are found everywhere including fingertips and bench tops, it is important to avoid these surfaces. Wear gloves. Do not touch the portion of sterile toothpicks, inoculating loops, the pipette tip, and agar plates that will come into contact with your bacteria. Transformation Occurs when a bacterium takes up and expresses a new piece of genetic material (DNA). Genes are often on a vector UV lamps Ultraviolet radiation can cause damage to eyes and skin. Short wave UV is more damaging than long wave (used in this experiment). Wear protective goggles. Vector An autonomously replicating DNA molecule into which foreign DNA fragments are inserted and then propagated in a host cell (a plasmid is a type of vector) 5 Equipment and Reagents *Record the procedure, observations, and answers to questions in the laboratory notebook *Wear gloves Reagents and equipment microcentrifuge tube racks and sterile microcentrifuge tubes p1000 pipettemen/tips p20 pipettemen/tips gloves small ice bucket/ice sterile toothpicks (autoclaved) long wave UV lamps 42 degree heat block 37 degree incubator LB broth (Luria broth) at room temperature CaCl2 (transformation) solution on ice, 50 mM 1 LB plate 2 LB/amp plates (0.1 mg/ml ampicillin) 1 LB amp/ara plate (0.1 mg/ml ampicillin + 6 mg/ml arabinose) Streaked plate of E. coli strain K12/HB101 pGLO plasmid DNA on ice 0.08 ug/ul stock solution Procedure Follow the Transformation Kit quick guide pg 14 - 15 Answer the following in the laboratory notebook 1. Why doesn’t the pGLO plasmid itself glow? 2. Why is pGLO referred to as a plasmid “vector”? 3. What is the role of the heat shock in transformation? 4. What are competent cells? 5. What is the role of ampicillin in the transformation process? 6. Why is the recovery period important? What would happen to cells if the recovery period was omitted and they were spread immediately onto LB amp plates? 7. Predictions: For each of the following, a – f, indicate if colonies will grow and if yes, will they glow? a. LB plate with non-transformed bacteria b. LB plate with transformed bacteria c. LB/amp with non-transformed bacteria d. LB/amp with transformed bacteria e. LB/amp/ara with non-transformed bacteria f. LB/amp/ara with transformed bacteria 8. What is the role of arabinose in the expression of GFP in the pGLO transformation system? 6 BIO208 Analysis of Transformation Results Record the following observations and analyses in the laboratory notebook. I. Analysis of plates 1. How much growth do you see on each plate, relatively speaking? If there are too many colonies to count use "TNTC" (too numerous to count). List all plates in the lab notebook and the growth or lack of growth observed on each. Write neatly, you can express the data in table form. 2. On which plate(s) did you expect to see NO bacterial growth and why? Do your observations support your hypothesis? Use full sentences and provide detail. 3. On which do you expect to see glowing bacteria and why? 4. Obtain the long wave UV light and examine the bacterial plates in the dark. Are there glowing bacteria? How many glowing colonies do you observe? 5. For each of the following scenarios consider the LB/amp/ara plate only *Discuss the expected outcome (what would be observed) *Assume that all other conditions are normal. * Provide clear and detailed explanations. a. b. c. d. e. E. coli cells are dead Arabinose is degraded Ampicillin is degraded Plasmid is degraded E. coli were contaminated with another, ampicillin-resistant bacteria II. Calculation of Transformation Efficiency Record the following observations and analyses in the laboratory notebook. The transformation efficiency (TE) is the extent to which the bacterial cells were genetically transformed. The TE is a # that represents the total # of bacterial cells that express the GFP divided by the amount (in ug) of plasmid DNA used in the experiment. Transformation efficiency = Total number of transformed cells on plate Amount of plasmid DNA spread on plate 1. Examine one LB/amp/ara plate under UV light. Each colony on the plate is composed of millions of cells (clones). Determine the number of individual colonies. Number of glowing colonies = 2. The concentration of plasmid DNA was ______ ug/ul (check with instructor). Look in your notes to find out the volume of pGLO used and then: 7 Calculate the total ug pGLO used in the transformation reaction = _______ ug 3. Determine the transformation efficiency. Use scientific notation to express the results (example 10,000 = 1X 105). Note the units = colonies/ug plasmid DNA Transformation Efficiency = _______________ colonies/microgram plasmid DNA (colonies/ug) 4. When making a bacterial library, scientists aim for transformation efficiencies over 2.5 X 10 6 colonies per microgram. To accomplish this, highly competent bacteria requiring extensive preparation are used. How does the transformation efficiency observed in this experiment compare? 5. Calculate. If an experiment had a transformation efficiency of 2 X 10 3 bacteria/ug DNA, how many transformant colonies would be expected to grow on the LB/amp/ara plate? (Assume 5 ul of pGLO at a stock concentration of 0.001ug/ul was used). 6. Based on your results in this experiment, what are your suggestions for future experimentation? Drawings or flow charts can be included in the experimental design description. Based on your observations, analyses, and conclusions what component or components should be changed or tested? If you obtained unexpected results, provide an explanation. *Plates are disposed of in the biohazard trash III. Pick colonies for GFP expression