Feeding a growing world: pglo transformation of E. coli

Transcription

1 : Transformation Feeding a growing world: pglo transformation of E. coli The issue The Earth s resources are limited, but the human population is growing fast. How can we ensure food security adequate safe, healthy food for everyone? Can we modify organisms quickly and safely to improve food security? How can we see easily which organisms have been successfully modified and which have not? The use of a plasmid vector, pglo, bearing a gene for a fluorescent protein is one answer to these questions. Introduction Key terms Aseptic technique: A protocol used in microbiology to prevent contamination by unwanted microorganisms and ensure you only grow the microbes that you want to investigate. Gene: A length of DNA that may code for one or more proteins or for a regulatory piece of RNA. Genetic transformation: A change caused by genes; the take-up and expression by a cell of a new piece of DNA, often producing a new visible characteristic in that organism. Nutrient medium: A medium for growing bacteria that provides the nutrient requirements of the bacteria. In this protocol the liquid nutrient medium (broth) and solid nutrient medium (agar) are both LB (Luria and Bertani), containing an extract of yeast and digested meat products, thereby providing carbohydrates, amino acids, nucleotides, salts and vitamins for bacterial growth. The solid medium can also contain ampicillin and arabinose, called LB/amp/ara agar. Phenotype: The observable characteristics of an organism. Plasmid: A small ring of DNA often found in bacteria in addition to their one large circular piece of DNA. These plasmids contain genes for traits that aid bacterial survival. In nature, bacteria may exchange plasmids, thus sharing these beneficial genes. This aspect of their physiology can be exploited in biotechnology to persuade bacteria to take up foreign DNA, transforming them. The plasmids are vectors. Recombinant DNA: A piece of DNA created by the combination of DNA strands from two different sources, such as a jellyfish gene inserted into a bacterial plasmid vector _pglo_ indd 43 06/04/ :00

2 : Transformation Vector (in biology): An organism or agent that acts as a carrier. In gene technology a vector carries and inserts foreign DNA into the host genome. Examples include plasmids, viruses and some bacteria. Use of the gfp gene In your first practical investigation, about reducing sugars in potatoes, you learned that some genetically modified (GM) varieties of potato have been developed. Your biology course covers the process of genetic modification, and in this session you will see how the gfp gene for green fluorescent protein (GFP) is used as a research tool. If the gene has been taken up it is expressed in the phenotype, and the protein can be seen under ultraviolet light as a green glow. Researchers can see whether a plant has been modified (if gfp has been taken up, so have the other genes in the plasmid) and can also see where exactly in the plant the genes are being expressed and whether different environmental conditions affect that expression. It is a widely used, safe and reliable research tool. The gene for GFP was originally obtained from the bioluminescent jellyfish Aequorea victoria. The gfp gene codes for GFP, a protein that absorbs UV light and re-emits it as green light (fluorescence). Many corals also contain this protein, which acts as a molecular sunscreen by absorbing UV light and re-emitting it at a longer, lower-energy wavelength, preventing damage to the organism by the UV light. The fluorescence cannot be seen during daytime, as sunlight is too bright, but it is visible when UV light is shone onto the organism in dim light. Did you know? Genetically modified fruit flies (Drosophila melanogaster) expressing GFP in their eyes. Derric Nimmo & Paul Eggleston / Wellcome Images The gfp gene has been around for over 160 million years in the jellyfish Aequorea victoria, which now lives in the cold waters of the north Pacific Ocean. Scientists in Sweden have harnessed a protein from Aequorea victoria to make miniature fuel cells for electronics. The gfp gene has been modified by a biotechnology company in California, Bio-Rad, to include specific mutations that enhance the fluorescence of the protein. In this practical protocol you will insert this gene into the bacterium Escherichia coli, which will enable you to visualise the genetic transformation as the bacteria express the gfp gene. You will be given the modified form of the gfp gene, inserted into Bio-Rad s pglo plasmid. When the bacteria take up this plasmid they express the gene, making the protein GFP that fluoresces very brightly, so you can see the green glow of the bacterial colonies when they are illuminated with a hand-held UV lamp or a UV pen light. Hence this protocol allows you to observe gene expression in real time. Practical investigation: pglo transformation of E. coli Aims In this investigation you will: move a gene (gfp) taken from a jellyfish into an E. coli bacterium using a plasmid as the vector find out how heat shock treatment helps the process _pglo_ indd 44 06/04/ :00

3 : Transformation Method understand the importance of using aseptic (sterile) technique develop an understanding of a technology that may help improve food security. Safety Carry out a risk assessment with your teacher. What hazards do you predict, and how will control them, that is, reduce the risks from them to a minimum? Ampicillin is a penicillin-type antibiotic. It is present in some of the agar plates in this practical investigation. If you are allergic to penicillin you should avoid any contact with ampicillin. The E. coli in this kit belong to the strain HB101 and are nonpathogenic. This strain is selected for work of this kind and must be grown in an enhanced nutrient medium, therefore it is not able to escape into the environment. However, you still need to use aseptic technique and observe normal safety procedures for carrying out practical investigations using microbes. This will reduce the chances of contamination of your plates (petri dishes), the lab and yourself, as well as teaching you best practice. Thoroughly disinfect work surfaces (which must be impervious) with disinfectant before and after carrying out the protocol. When taping plates shut it is important to use just two pieces of tape, one on each side of the plate. Do not seal the plate completely, as this creates an anaerobic environment, which can allow dangerous microorganisms to grow, and allows drops of condensation to form, which can carry microorganisms out of the plate. Report spillages to your teacher/technician. Follow your teacher s instructions about disposal of material (once material is collected, it must be autoclaved before disposal). Cover all cuts. Wash your hands with soap before and after carrying out the protocol and before leaving the lab. No eating, drinking or applying cosmetics. Take care when handling disinfectant solution it can damage skin. Work in such a way as to minimise the production of aerosols. UV light can damage your eyes. Ensure that you point the UV source downwards and away from yourself and other people, and do not look into it _pglo_ indd 45 06/04/ :00

4 : Transformation Equipment 1 E. coli starter plate (LB) 4 poured agar plates (1 LB, 2 LB/amp, 1 LB/amp/ara) 1 ml (cm 3 ) transformation solution (sterile calcium chloride, CaCl 2, solution) 1 ml LB nutrient broth 7 sterile inoculation loops 5 sterile micropipettes 2 microcentrifuge tubes 1 foam microtube holder 1 polystyrene cup full of crushed ice 1 marker pen timer sticky tape container of disinfectant solution for disposal of used pipettes and loops laminated paper mat pre-soaked in disinfectant solution Access to: rehydrated pglo plasmid DNA water bath at 42 C UV light incubator set at 37 C (or 28 C or 21 C see step 17 of procedure) 1. Microbiological techniques If you have not carried out any microbiological practicals before you will spend one lesson on basic microbiological techniques (aseptic technique, preparing plates, plating out and using micropipettes). These techniques for example never putting a loop down on a surface, disinfecting the work surface before and after work are designed to ensure that no microbes from the environment can get onto a plate, and no microbes from your work can escape into the environment. In your next lesson you will carry out the genetic transformation. Note that although in this protocol you open the starter plate in order to transfer a colony of E. coli, you should normally leave all plates taped shut. 2. The transformation Bio-Rad s pglo plasmids contain: the gfp gene a gene (bla) that codes for the enzyme beta-lactamase, which confers resistance to the antibiotic ampicillin a gene regulation system that can control the expression of the gfp gene, which directs the synthesis of GFP, in the transformed bacterial cells. In order to get the modified plasmids into the bacterial cells, you will: treat the bacterial cells with transformation solution (calcium chloride) subject the bacteria to heat shock. The calcium ions neutralise the negative charges of the phosphate groups in the DNA backbone and in the phospholipids of the cell surface membrane. This reduces the normally repulsive forces _pglo_ indd 46 06/04/ :00

5 : Transformation between the DNA of the plasmid and the cell surface membrane of the bacterial cells, allowing the plasmids to enter the cells. Heat shock increases the permeability of the cell surface membrane to the DNA of the plasmid. 3. Ampicillin resistance To enable the transformed cells to grow in the presence of ampicillin you will: provide them with nutrients, including arabinose incubate them at a suitable temperature long enough for them to start expressing their newly acquired gene and synthesise the GFP. When ampicillin is included in the nutrient agar it kills most E. coli only transformed bacteria (those that have taken up the plasmid) can grow, as only the transformed bacteria contain a plasmid that includes a bla gene to confer ampicillin resistance. When the sugar arabinose is added to the nutrient medium it switches on the gfp gene in transformed bacteria, so they can make the GFP and will glow green under UV light. Transformed bacteria grown on nutrient agar without arabinose will appear white. 4. Results and further work In a follow-up lesson you will observe the results and assess the success of your work, as well as discussing some of the applications and uses of the gfp gene in real life and considering the use of GM animals and plants. Did you know? E. coli bacteria are part of your gut microbiota. We each have many species of bacteria and Archaea living inside our gut lumen (so not inside our body tissues). These organisms help us to digest food and produce some vitamins, hormones and other chemicals that help regulate appetite and other aspects of our metabolism. We may have more microbial cells in the gut, mouth and vagina and on the skin than we have cells in our body. However, since prokaryotic cells are much smaller than eukaryotic cells, the total mass of the microbiota of a human is estimated to be as much as 2000 g, or 1 3% of body mass. Repeated or prolonged exposure to antibiotics may disrupt the balance of the types of microbes within the gut microbiota. Such disruptions could lead to a person not being able to properly regulate appetite and could contribute to obesity. Similarly, overexposure to antibiotics may upset the balance of microbes in the gut such that one type, Clostridium difficile (C. diff) is no longer kept in check and can cause an infection, leading to persistent and severe diarrhoea. This infection can be fatal and is often very difficult to treat. However, faecal transplants (faeces from a healthy donor) into the colon of the recipient are proving effective in 90% or more of cases. The donated faeces has to be screened for pathogens first _pglo_ indd 47 06/04/ :00

6 : Transformation Figure 2 Dispersing colonies in a solution in a microtube. Step-by-step procedure 1. Ensure that your group has all the equipment listed before you start. Wash your hands. Work over a disinfected, impervious surface. Label one closed micro test tube and label the other pglo. Label the tubes with your group s name and place them in the rack. 2. Open the tubes and use a sterile transfer pipette to transfer 250 µl (0.25 ml) of transformation (calcium chloride) solution into each tube. 3. Place the tubes (still open and still in the rack) on crushed ice. 4. Using a sterile loop pick up 2 4 large colonies of bacteria from your starter plate. The bacteria in colonies are actively dividing, hence actively growing. Choose colonies that are uniformly circular and have smooth edges. Pick up the tube and immerse the loop into the transformation solution. Spin the loop between thumb and index finger until the entire bacterial colony is dispersed in the transformation solution (Figure 2). Place the tube back on the ice. Drop the loop into the disinfectant solution (it will be disposed of later). 5. Now use another sterile loop and repeat step 4 for the pglo tube. 6. Examine the solution of pglo plasmid DNA under the UV light. Note your observations. 7. Immerse a new sterile loop into the stock tube of rehydrated plasmid DNA. Withdraw a loopful and make sure that there is a film on the loop (it should resemble the soapy film you get when using soap solution to blow soap bubbles). Mix this loopful into the cell suspension of the tube. Alternatively, use a new sterile micropipette to transfer 10 µl (0.01 ml) plasmid solution. Again, drop the loop or micropipette into the disinfectant. 8. Close both tubes (the and the pglo ) and leave them both on ice for 10 minutes. Make sure the tubes are pushed all the way down into the foam rack so their bases are in the crushed ice. 9. During this 10 minutes label your four agar plates, on their bases, not their lids, as follows. The agar type will already be labelled: pglo on LB agar/your group name pglo on LB/amp agar/your group name on LB/amp agar/your group name on LB/amp/ara agar/your group name. 10. Double-check the temperature of the water bath with the thermometer. It should be 42 C. At the end of the 10 minutes, transfer the foam rack with the two tubes from the ice into the water bath at 42 C for exactly 50 seconds. This is the heat shock treatment. 11. Then place both tubes, in their rack, back on the ice for 2 minutes. The times and temperature here are critical. The rapid transfer from ice to heat and back to ice gives the heat shock, and any time less or greater than 50 seconds at 42 C reduces the ability of the bacteria to take up plasmids. 12. After 2 minutes remove the rack and tubes from the ice and place on your bench _pglo_ indd 48 06/04/ :00

7 : Transformation Wash your hands and disinfect the surface. 3 Add 0.25 ml transformation solution to each tube. 1 ml 750 µl 500 µl 250 µl 100 µl 4 Place open tubes on crushed ice. Transfer 2 4 starter colonies into tube with a loop. 2 Label closed micro tubes,. 1 ice 250 µl transformation solution 6 With a fresh loop, repeat step 4 for tube. 7 Examine the pglo plasmid DNA N NA solution under UV. 8 Transfer 0.01 ml pglo plasmid into tube with a new loop. plasmid DNA 9 10 Label your four agar plates with your group name. 11 Check that water bath is at 42oC. Warm tubes for 50 s. ice Close tubes, push their bases into the ice and leave for 10 min. 5 rack ice 12 Put tubes and rack on your workbench. Place tubes back on ice for 2 min. ice water bath 42oC for 50 sec 14 With a sterile pipette, add 0.25 ml LB broth to With a new pipette, add 0.25 ml LB broth to. 15 Pipette 0.1 ml of the correct suspension to each plate. Use a fresh sterile pipette each time. 100 µl 17 Streak each suspension over its plate with a new loop. 18 Tape the plate closed, stack them and incubate them at 37 C for 24 h. 19 Disinfect your work surface and wash your hands. LB/ampLB/amp/ara LB/amp LB on LB agar on LB/amp agar on LB/amp agar on LB/amp/ara/agar 250 µl LB-broth 16 Incubate both tubes for 10 min at room temperature _pglo_ indd 49 06/04/ :00

8 : Transformation 13. Open the + pglo tube, use a new sterile pipette to add 250 µl (0.25 ml) of LB nutrient broth to the tube and then reclose the tube. 14. Using a new sterile pipette, repeat step 13 for the pglo tube. 15. Incubate both tubes for 10 minutes at room temperature. This allows the bacteria time to recover and to express the beta-lactamase enzyme that gives it resistance to ampicillin, so that transformed bacteria can survive on the LB/amp and LB/amp/ara plates. 16. Now gently flick the closed tubes with your finger to mix the contents within each tube. Using a new sterile pipette for each tube, pipette 100 µl (0.1 ml) from each of the tubes onto the corresponding plates. 17. Use a new sterile loop for each plate and spread the suspensions evenly over the surface of the agar jelly. To do this quickly skate the flat surface of the loop back and forth and criss-cross over the agar surface (Figure 3). Figure 3 Streaking a plate. 18. Replace the lids on the agar plates and tape them in two places, then stack the plates and tape them with a different-coloured tape, as shown in Figure 4. Incubate the plates upside down (this prevents any condensation from running onto the bacterial colonies) in the incubator set at 37 C for 24 hours (or 28 C for 48 hours or 21 C for 72 hours, depending on when your next lesson is). 19. Leave your disposable swabs or loops and micropipettes soaking in the disinfectant and thoroughly disinfect your work surface. When you have finished, wash your hands. Figure 4 Agar plates labelled, taped closed, stacked upside down and taped together. 20. Before you observe your incubated plates, predict what you expect to see. (a) On which plate(s) would untransformed E. coli bacteria grow? Explain your prediction. (b) On which plate(s) would transformed E. coli bacteria grow? Explain your prediction. (c) On which plate(s) would bacterial colonies glow green under UV light? Explain your prediction. 21. Observe the colonies on your incubated plates, without and with UV light. Record your observations by drawing what you see on each plate and adding notes on your observations. Compare the relative bacterial growth on each plate. Record the colour of the colonies and record the number of colonies on each plate _pglo_ indd 50 06/04/ :00

9 : Transformation Questions 1. Some of your bacteria (those that have been transformed) glow green under UV light. There are two possible sources of this fluorescence the gfp gene and the protein GFP. How can you tell that it is not the gene that directly fluoresces? 2. Explain your observations recorded in stage 20 above. 3. For an organism to be genetically transformed, a copy of the new gene has to be inserted into each of its cells. Explain why populations of bacteria are well suited to being totally genetically transformed. 4. Suggest why bacteria are useful organisms to enable scientists to see if the new gene (or trait) is passed to subsequent generations. 5. Why is the gfp gene a useful gene for allowing scientists to see from the phenotype whether the bacteria have taken up this gene and become transformed? 6. An organism s traits (phenotype) are usually caused by a combination of its genes (genotype) and its environment. Which two factors in the environment must be present for you to see the green fluorescence of the transformed bacteria? 7. In most organisms genes can be turned on or off in response to certain conditions. (You may have learned about the lac operon.) What is the advantage to an organism in being able to switch genes on or off? Did you know? The hundreds of different species of microbes in the human gut together have more genes than are present in the human genome. Many of these microbial genes encode proteins that we use and rely on for healthy functioning of our metabolism. Some scientists say we should therefore consider ourselves to be superorganisms. Other examples of superorganisms include bee colonies, ant colonies and termite colonies. For most of us our gut is colonised with microbes at birth. Babies are born head-down so they pick up microbes from the mother s anus. They will also take in microbes from the mother s skin during suckling and cuddling, and from contact with other surfaces. Babies born by caesarean section do not acquire the mother s gut microbes at birth so their gut microbiota take a little longer to become established. Certain foods, such as live yoghurt, are recommended because they contain friendly or good bacteria that we need in our gut. However, if you do not eat a healthy diet, these bacteria will not thrive. Diets with lots of vegetable matter, such as leeks, promote the growth of the bacteria we need in our gut _pglo_ indd 51 06/04/ :00

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