Feeding a growing world: pglo transformation of E. coli

16 19 2: 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, to insert a gene for a fluorescent protein is one answer to these questions. Introduction Food security and genetic modification The teacher notes for the first practical investigation in this topic introduce the issue of food security. Human population growth and increasing demand for meat, climate change and changing land use, are all factors that could threaten human health and food security. Humans depend upon only a few species of staple crops, including wheat, quinoa, sorghum, millet, rice, maize and potatoes. Most were developed by natural hybridisation and polyploidy followed by thousands of years of selective breeding, a somewhat hit-or-miss process. Now scientists have the knowledge and technology to modify crop plants in a more exact way with genetic modification. In this investigation students will have the opportunity to try some techniques used in genetic modification, using the gfp gene for green fluorescent protein (GFP), and to explore the pros and cons of this technology. In the additional activities on The Crunch website, thecrunch. wellcome.ac.uk/schools other, less high-tech methods of increasing food production and availability will be considered, along with GM technology and its risks and benefits. Tools for genetic modification GFP has existed in jellyfish for over 160 million years. Solutions of purified GFP appear yellow under room light, but when exposed to sunlight the protein absorbs UV light of wavelength 395 nm and emits light of 509 nm as a green glow. GFP was first discovered and studied in the 1960s and 70s by Osamu Shimomura. However, its usefulness as a research tool was not realised until 1992. In 2008 Shimomura shared the Nobel Prize in Chemistry with Martin Chalfie and Roger Y Tsien. You will find additional notes on the protein and its uses in More to explore on The Crunch website. 27 23_Teacher_notes_16-19_pglo_027-034.indd 27 06/04/2016 16:59

16 19 2: Transformation The practical investigations The gfp gene is used as a research tool; if it has been taken up it is expressed in the phenotype and the protein can be seen with the use of ultraviolet light as a green glow. Researchers can thus see whether the organism has been modified (if gfp has been taken up, so have the other genes) and can also see where exactly in the organism the genes are being expressed and whether different environmental conditions affect that expression. It is a widely used, safe and reliable research tool. This practical investigation involves the transformation of E. coli bacteria by inserting the gfp jellyfish gene. The straightforward protocol can be carried out in one lesson, with a second lesson to examine the bacteria, and will enable students to visualise some of the steps involved in genetic modification of organisms. It will also stimulate debate about the use of genetically modified organisms (GMOs), and follows on from the first set of activities, in which the potentially harmful effect of high sugar content in potatoes was investigated and the idea of modifying potatoes to have a lower sugar content was introduced. Derric Nimmo & Paul Eggleston / Wellcome Images GM fruit flies (Drosophila melanogaster) expressing GFP in their eyes. Research informing the investigation Professor Rod Scott, Head of the Department of Plant Genetics at the University of Bath, UK, and his team have research interests that include the use of genetic modification to create transgenic plants for bioethanol production. Some current aspects of research into food security and the factors that put it at risk are considered in the references at the end of this set of teacher notes and in the additional links provided on The Crunch website, along with references on research related to plants and jellyfish genes. Rothamsted Research Professor Jonathan Napier at Rothamsted Research has provided a short video on his work on Making fish oils in plants, which can be accessed on The Crunch website, thecrunch.wellcome.ac.uk./schools Genetically modified Camelina sativa produces oils used to feed fish. 28 23_Teacher_notes_16-19_pglo_027-034.indd 28 06/04/2016 16:59

16 19 2: Transformation Assumed prior learning cell structure eukaryotic and prokaryotic; cell division in prokaryotes; plant cell structure including cell walls and middle lamella carbohydrate molecular structures; hydrolysis, condensation reactions; protein structure, protein synthesis enzymes; digestion of nucleic acids nuclease and nucleotidases microbiological techniques: aseptic technique, streak plating, and general safety rules for such practicals DNA, RNA and biotechnology. Learning objectives Use microbiological aseptic techniques, including the use of agar plates and broth. Develop understanding of the use of plasmid vectors in transforming organisms. Discuss the role of genetically modifying crops to help meet the increased demand for food during the 21st century. Visualise the process of genetic modification and see how the gfp gene works as a research tool. Understand that the gfp gene codes for the protein GFP and be able to deduce that the protein and not the gene fluoresces. Recall the structure of prokaryotic cells, including plasma membrane cell wall and plasmids. Demonstrate understanding of the ethical issues surrounding use of GM organisms. Activities Aim In this investigation students 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 understand the importance of using aseptic (sterile) technique develop an understanding of a technology that may help improve food security. Introductory activities A presentation, pglo transformation of E. coli, and a video of the Bio- Rad experimental protocol, Bacterial transformation, (available on The Crunch website, thecrunch.wellcome.ac.uk/schools) introduce the use of the gfp gene as a marker in genetic modification. Students should be given the student activity sheet to read as a homework task before the practical lesson. If students are unfamiliar with microbiological techniques, a preliminary lesson introducing them to aseptic technique, preparing agar plates (petri dishes), plating out and using micropipettes will be necessary. 29 23_Teacher_notes_16-19_pglo_027-034.indd 29 06/04/2016 16:59

16 19 2: Transformation Practical investigation: pglo transformation of E. coli Safety Carry out a risk assessment with the class. What hazards do you predict, and how will you control them? Check for penicillin allergies. Ampicillin is a penicillin-type antibiotic. It may cause irritation to eyes or the respiratory system. In the case of contact with eyes, rinse immediately with plenty of water and then seek medical advice. Anyone allergic to penicillin or related compounds should avoid contact with ampicillin. The E. col in this kit belong to the strain HB101 and are non-pathogenic. 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, students must nevertheless use aseptic technique and observe normal safety procedures for carrying out practical work using microbes. This will reduce the chances of contamination of their plates, the lab and themselves, as well as teaching them best practice. Thoroughly (15 minutes) disinfect work surfaces (which must be impervious) with 1% Virkon disinfectant solution before and after carrying out the protocol. Report spillages. Follow instructions about disposal of material (once material is collected, it must be autoclaved before disposal). Cover all cuts. Wash hands with soap before and after carrying out the protocol and before leaving the lab. No eating, drinking or applying cosmetics. Work in such a way as to minimise the production of aerosols. Plates will be taped shut at opposite sides as the students inoculate them. They should be checked after the lesson and if necessary taped more securely (while still permitting air to circulate) so that they cannot be reopened. Students should not open the plates to observe the transformed bacteria. Schools should not usually incubate cultures above 30 C unless microbial growth is very limited at lower temperatures. Since in this case a temperature of 37 C is used, great care must be taken to ensure that plates are not opened and that they are disposed of correctly. If there is more than one day between lessons, the plates can be incubated at a lower temperature, to slow the growth of the bacteria (see the instructions in the student activity sheet). If necessary, incubated plates can be stored between lessons, upside down so that any condensation runs onto the lids and not the bacteria, in the fridge at 4 C. Only staff should have access to the plates. Students should label their plates on the underside, not on the lids. After plating out/streaking, the lids must be replaced and taped with two pieces of tape. Each group s set of four plates can be stacked up and taped together again with different-coloured tape for incubation (see the diagram in the student activity sheet). For observation the second tape will need to be removed, but not the tapes keeping the lids in place. The plates (including unused ampicillin agar plates) should be disposed of safely afterwards by the technician, autoclaving and then bagging for disposal. The UV source is a long-wavelength source (315 400 nm). Students should be reminded to point the source downwards and away from people. UV-rated goggles or sunglasses are not necessary provided that the light is not observed directly. Hard UV lamps must not be used. 30 23_Teacher_notes_16-19_pglo_027-034.indd 30 06/04/2016 16:59

16 19 2: Transformation CLEAPSS G272: Gene technology, a starter guide to health and safety gives details of the legal regulations governing this activity, and a guide to The Genetically Modified Organisms (Contained Use) Regulations 2014 can be downloaded from the general publications section of the CLEAPSS website, cleapss.org.uk. The regulations covering work with GMOs are also described in the Association for Science Education document Topics in Safety, Topic 16: Working with DNA (http:// www.ase.org.uk/documents/hands-resource-topics-in-safety-16-working-with-dna/topic-16- working-with-dna-revised2014-.pdf). In Scotland, schools must follow the guidance in Materials of Living Origin Educational Uses: A Code of Practice for Scottish Schools and Colleges and Safety in Microbiology: A Code of Practice for Scottish Schools and Colleges. These are available for free download from the SSERC website. In England, Wales and Northern Ireland, consult the current advice on microbiology on the CLEAPSS website. Material safety data sheets (MSDSs) are available on the Bio-Rad website, www.bio-rad.com. This protocol was developed by Bio-Rad (www.bio-rad.com/en-uk/ category/pglo-plasmid-gfp-kits) for use in schools and colleges. The strain of E. coli used is non-pathogenic. The practical works well in one practical session with one follow-up session for results and discussions. Bio-Rad s pglo plasmids contain: the gfp gene that codes for green fluorescent protein (GFP) 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 GFP in the transformed bacterial cells, switching it on when arabinose is present. In the first lesson, students transform E. coli taken from a starter plate that has been prepared in advance. They treat the E. coli with CaCl 2 and heat shock, then use pglo plasmids to introduce genes including gfp and bla (for ampicillin resistance). Students inoculate several agar plates with their sample (+pglo) and control ( pglo). The transfer of E. coli colonies from the starter plates to sterile plates requires that the starter plate be reopened after incubation. Students should be reminded that plates are never normally opened once inoculated. The plates are incubated before the second lesson, in which students observe that the transformed E. coli have grown even in the presence of ampicillin, and fluoresce under UV light if they have a source of the sugar arabinose. The students sample and control plates are: pglo on LB agar pglo on LB/amp agar +pglo on LB/amp agar +pglo on LB/amp/ara agar. 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. 31 23_Teacher_notes_16-19_pglo_027-034.indd 31 06/04/2016 16:59

16 19 2: Transformation When the sugar arabinose is included in 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. Detailed instructions including equipment, safety information and method are in the technician notes and the student activity sheet. In the second lesson, students will observe colonies on the incubated plates and assess the success of the 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. Additional activities Attitudes to genetic modification: case studies A series of case studies of genetic modification and its consequences are provided on The Crunch website, thecrunch.wellcome.ac.uk/ schools, for discussion. Attitudes to genetic modification: activities survey debate case study on the Scottish Parliament decision to prohibit the growth of GM crops. Students could use the information given in the case studies to inform their ideas and develop suitable short surveys. They could administer these to others in their year, and friends and family members. All participants responding to the survey should be over 16 years of age so they can give informed consent to taking part. The surveys should be short for ease of response and analysis. Students could formulate a large question to investigate, such as: does the acceptance of GM for non-food organisms differ from that of GM food? do people appreciate that food security may be uncertain, and what options for improving food security would they be prepared to accept? how widespread are misconceptions about GM? A set of topical play scripts and supporting resources from the Theatre of Debate is available on The Crunch website: The Fat of the Land Adam Hughes The Super Safe Environment Compound Elinor Roos Fields and Fields and Fields Jonathan Hall The Chicken Temptation Judith Johnson Feed Me Rhiannon Tise. 32 23_Teacher_notes_16-19_pglo_027-034.indd 32 06/04/2016 16:59

16 19 2: Transformation Answers to questions in student activity sheet Step-by-step procedure: examining the incubated plates 20. (a) pglo LB plates, as untransformed bacteria do not have the ampicillin resistance gene. (b) +pglo LB/amp and +pglo LB/amp/ara, as only these bacteria have been exposed to the plasmid, and those that have taken up the plasmid will have an ampicillin resistance gene enabling them to grow on these media. (c) +pglo with LB/amp/ara as arabinose is need to switch on the gfp gene. 21. Drawings and descriptions of students observations and findings. Answers to questions in student activity sheet 1. Because the pglo plasmids did not fluoresce/glow green when examined under UV light. 2. Colonies of transformed bacteria grow on the +pglo LB/amp plate as they have the gene for the enzyme beta-lactamase, giving ampicillin resistance. However, with no arabinose sugar present, the gfp gene is not switched on so these colonies do not glow green under UV light. Colonies of transformed bacteria grow on the +pglo LB/amp/ ara plate as they have the gene for the enzyme beta-lactamase, giving ampicillin resistance. With arabinose also present in the medium, the gfp gene is switched on and these cells produce GFP. This protein absorbs UV light of wavelength 395 nm and emits light of wavelength around 500 nm (green). On the other two plates, bacteria are not transformed as they have not been mixed with plasmids. No E. coli will survive on the pglo LB/amp plate, as they are untransformed so have no ampicillin resistance. Untransformed E. coli will grow on the pglo LB plate, but they will not fluoresce under UV light. 3. Each organism is a single cell, and each gives rise to individual single cells when reproducing by binary fission. As binary fission is asexual, all individuals arising from a transformed bacterium are clones of that original bacterium, that is, genetically identical to it. When bacteria divide, their plasmids, as well as their main DNA, duplicate and so each daughter cell receives a copy of all the cell s DNA. 4. Their generation time is short 30 minutes in some cases so several generations can be observed in a short period. 5. Because it glows green under UV light so can easily be observed. 6. Ampicillin and arabinose in the growth/nutrient/culture medium. 7. Avoids wastage of resources (ATP/energy hence respiratory substrate, and amino acids) in making enzymes that are not needed. 33 23_Teacher_notes_16-19_pglo_027-034.indd 33 06/04/2016 16:59

16 19 2: Transformation Research background Additional web resources on this topic will be found as links on thecrunch.wellcome.ac.uk/schools. Do we need more food or do we simply waste too much? Food security, threats, and agricultural techniques: Lynas, M. The God species how humans really can save the planet. Fourth Estate, 2012 Ronald, P, and Adanchak, R. Tomorrow s Table Organic Farming, Genetics and the Future of Food. OUP, 2009 Future Food, including section on sustainability and technology futurefood2050.com Food Climate Research Network (FCRN) including a library of research www.fcrn.org.uk/ UN Food and Agriculture Organization: Climate-smart agriculture www.fao.org/climate-smart-agriculture/72616/en/ Genetic modification and GFP: Professor Rod Scott, Head of the Department of Plant Genetics at the University of Bath www.bath.ac.uk/bio-sci/contacts/academics/rod_scott/ Sense about Science on GM (the site also covers other food-related subjects) www.senseaboutscience.org/subjects.php?action=tag&id=56 Pieribone, V, Gruber, D.F. Aglow in the Dark, The Belknap Press, Harvard University Press, 2005 Zimmer, M. Glowing Genes, Prometheus Books, 2005 Movie showing a cell producing GFP-tagged HIV particles: www.nature.com/nature/journal/v454/n7201/suppinfo/nature06998.html Parliamentary briefings on GM topics: www.parliament.uk/business/publications/research/briefing-papers/post-pn-482/gm-cropsand-regulation www.parliament.uk/business/publications/research/briefing-papers/post-pn-412/gm-inagricultural-development www.parliament.uk/business/publications/research/briefing-papers/post-pn-483/gm-insectsand-disease-control 34 23_Teacher_notes_16-19_pglo_027-034.indd 34 06/04/2016 16:59