NATIONAL QUALIFICATIONS CURRICULUM SUPPORT Biology Unit 2, Part 3: Metabolism in Microorganisms Teacher’s Notes [HIGHER] The Scottish Qualifications Authority regularly reviews the arrangements for National Qualifications. Users of all NQ support materials, whether published by Learning and Teaching Scotland or others, are reminded that it is their responsibility to check that the support materials correspond to the requirements of the current arrangements. Acknowledgement Learning and Teaching Scotland gratefully acknowledges this contribution to the National Qualifications support programme for Biology. © Learning and Teaching Scotland 2011 This resource may be reproduced in whole or in part for educational purposes by educational establishments in Scotland provided that no profit accrues at any stage. 2 UNIT 3, METABOLISM AND SURVIVAL (H, BIOLOGY) © Learning and Teaching Scotland 2011 Contents (b) Genetic control of metabolism (i) Genetic variation 4 4 (b) Genetic control of metabolism (ii) Recombinant DNA technology 7 7 (c) Ethical considerations in the use of microorganisms UNIT 2: METABOLISM IN MICROORGANISMS (H, BIOLOGY) © Learning and Teaching Scotland 2011 11 3 METABOLISM IN MICROORGANISMS Investigating metabolism in microorganisms (b) Genetic control of metabolism (i) Genetic variation Links to prior/prerequisite knowledge SCN 3-14b SCN 4-14c Students have already studied mutations in Unit 1 (see Section 3, The Genome) and so should be aware that physical changes to the DNA of a cell or a change in the number of chromosomes can arise naturally. They should also be familiar with the concept of improvement through mutat ion since polyploidy crops are discussed in Unit 1. New content areas Examples of mutagenic agents and their effect on genetic material. Transfer of DNA between bacteria, uptake of DNA by bacteria from their environment. Production of new genotypes by sexual reproduction between existing strains of fungi and yeast. Background information Mutations may arise naturally by physical change s to the DNA of a cell or a change in the number of copies of an entire gene or chromosome. When such a change in genotype produces a change in phenotype, the organism affected is called a mutant. In natural conditions, mutations arise spontaneously and at random. While they occur rarely, the frequency of mutation can be increased by exposure to mutagenic agents such as mustard gas and various types of radiation. Exposure to natural mutagens such as ultraviolet (UV) light, to industrial or environmental mutagens such as benzene or asbestos can all cause mutations. For geneticists, the study of mutagenesis is important because mutants reveal the genetic mechanisms underlying heredity and gene expression. Genetic transformation is the uptake of DNA from the environment. The cell is genetically altered as a result of direct uptake, incorporation and expression of DNA from its surroundings. Transformation occurs most commonly in bacteria and in some species occurs naturally. Bacteria capable of being transformed are said to be competent. Transformation is thought to be a significant cause of increased drug resistance when one bacterial cell acquires resistance and quickly transfers the resistance genes to many other cells. The 4 UNIT 2: METABOLISM IN MICROORGANISMS (H, BIOLOGY) © Learning and Teaching Scotland 2011 METABOLISM IN MICROORGANISMS main method of cell-to-cell transfer is conjugation. Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell -to-cell contact or by a bridge-like connection between two cells. During conjugation the donor cell provides a genetic element that is most often a plasmid. The plasmid transferred is often beneficial to the recipient. Benefits may include antibiotic resistance. A fungus is a member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and moulds. Although many species of fungi and yeasts reproduce asexually, many also carry out sexual reproduction and therefore have the ability to increase genetic variation. During this process, meiosis forms genetically varied spores that are then released from the fungi by specialised mechanical or physiological mechanisms. Identification of key concepts Mutations are rare but their incidence can be increased by exposure to mutagenic agents. Mutagenic agents can be chemical, such as asbestos or mustard gas, physical , such as several forms of radiation, or biological, such as bacteria phage. Mutations may be of benefit to the species or may introdu ce characteristics of commercial value. Recombinant DNA technology allows deliberate alteration of a genome. This may involve the addition, modification or deletion of one or more genes in a cell. As a result the cell may receive an additional property, fo r example the ability to make a new protein. Some species of bacteria are able to carry out transformation that involves uptake of DNA from their environment or another cell. The most common form of transformation is called conjugation and involves direct contact between two cells. DNA can then be transferred from a donor cell to a recipient cell. Many species of fungi are able to reproduce sexually and therefore increase variation. This is most commonly achieved by the production of genetically varied spores that are dispersed from the fungi and fuse with other sexual spores. Identification of particular areas of difficulty The idea of sexual spores produced by fungi is probably a new concept and many available sources of information on this topic are very advanced and involve challenging vocabulary. UNIT 2: METABOLISM IN MICROORGANISMS (H, BIOLOGY) © Learning and Teaching Scotland 2011 5 METABOLISM IN MICROORGANISMS Links to websites, animations, PowerPoints, audio or video files etc http://www.bbc.co.uk/learningzone/clips/mutations-and-geneticdiseases/10653.html http://www.microbiologyonline.org.uk/about -microbiology/introducingmicrobes/fungi http://highered.mcgrawhill.com/sites/dl/free/0072835125/126997/animation6.html http://www.microbeworld.org/index.php?option=com_content&view=article&id= 123&Itemid=118 http://www.bbc.co.uk/learningzone/clips/genetic-engineering-and-insulinproduction/4200.html Co-operative Learning Activities 3 and 4 Other useful information to stimulate interest 6 UNIT 2: METABOLISM IN MICROORGANISMS (H, BIOLOGY) © Learning and Teaching Scotland 2011 METABOLISM IN MICROORGANISMS (b) Genetic control of metabolism (ii) Recombinant DNA technology Links to prior/prerequisite knowledge Refer to Organisation of DNA in prokaryotes and eukaryotes in Unit 1 to remind students about the existence of circular chromosomes. Students will probably be aware of the use of genetic engineering for the production of insulin, factor VIII and human growth hormone, and should therefore be familiar with the role of plasmids and the process at a basic level. New content areas Use of recombinant DNA technology to create enzymes, genetically modified foods and pharmaceuticals and its future role in gene therapy. Examples of species commonly used for genetic engineering, such as E. coli, and reasons for their suitability. When the gene for a protein is cloned, it is placed on a plasmid adjacent to a region where the expression of genes can be controlled easily. Structure of plasmids/vectors. Use of endonucleases and ligase during the process of genetic engineering . Yeast as an alternative to bacteria. Background information The field of genetic engineering involves the isolation, manipulation and expression of genetic material. Genetic engineering is a rapidly growing technology and it is thought that it will have profound effects on our everyday lives. Some examples of how it may affect us are: - In the field of medicine it may improve the diagnosis and cure of hereditary defects and disease. - It is being used for the development of new drugs and vaccines for use by humans and animals. - In agriculture it is being used to improve food production. - It is being used to monitor and reduce environmental pollution. The process commonly utilises bacterial cells and their plasmids. Foreign genes can be inserted into isolated plasmids , which are returned to the bacterial cells. The cells reproduce, cloning the recombinant DNA as the cells replicate their plasmids. Under suitable conditions , the bacterial culture will produce the protein encoded by the foreign gene. Genetic engineering requires three biological ‘tools’. 1. Enzymes to cut DNA: The first step in many genetic engineering processes is the isolation of DNA from cells. When purified DNA has UNIT 2: METABOLISM IN MICROORGANISMS (H, BIOLOGY) © Learning and Teaching Scotland 2011 7 METABOLISM IN MICROORGANISMS been obtained it is cut into smaller fragments using restriction endonucleases. These enzymes were discovered in the late 1960s and work by cutting up DNA. They recognise and cut short specific sequences (between four and eight base pairs) within DNA. One of the most commonly used restriction enzymes is called EcoR1. It recognises the following six-base pair DNA sequence: 5′ GAATTC 3′ 3′ CTTAAG 5′ EcoR1 then cuts the DNA sequence as follows: 5′ G 3′ CTTAA AATTC 3′ G 5′ When EcoR1 cuts DNA it produces two double -stranded fragments, but the cuts do not occur at the same position. Instead the cut is staggered by four nucleotides, so that the DNA fragments have single -stranded overhangs (known as sticky ends). If another piece of DNA is cut with the same enzyme and so has the same sticky ends, the pieces of DNA can be joined together by base pairing between the sticky ends. Genes of interest and suitable vectors are treated with the same endonucleases to create complementary sticky ends, which are then combined using DNA ligase to form recombinant DNA. 2. A vector or transfer agent such as a plasmid : Cloning vectors can be manipulated so that they have the following characteristics: (a) They can be cut with restriction enzymes and foreign DNA sequences (cut with the same restriction enzymes) can be inserted into them using an enzyme called DNA ligase. (b) Antibiotic resistance marker genes can be added to them. These genes code for proteins that break down antibiotics. If a cloning vector is inserted into a microorganism, the microorganism gains the antibiotic resistance gene and so is able to grow in the presence of this antibiotic. The microorganism becomes resistant to the antibiotic and can be easily identified. (c) Some cloning vectors contain part of the lac operon. This is used to control the expression of the foreign DNA sequences. The foreign DNA is transcribed and translated only when the lac operon is switched on. After a foreign sequence of DNA has been inserted into a cloning vector using DNA ligase, the cloning vector is mixed with the microorganism into which it is to be transformed. Some of the 8 UNIT 2: METABOLISM IN MICROORGANISMS (H, BIOLOGY) © Learning and Teaching Scotland 2011 METABOLISM IN MICROORGANISMS microorganisms will take up the cloning vector, some will not. To separate the transformed microorganism from those that are not, it is grown in media containing the antibiotic to which the transformed microorganism has acquired resistance. The transformed microorganism has the cloning vector that has the antibiotic resistance gene, so it is able to grow in the presence of the antibiotic. Any microorganism that does not possess the cloning vector is unable to grow in this medium. The transformed microorganism is isolated from the medium and transferred to another medium where it is allowed to reproduce and grow in large quantities. Each new microorganism that is produced is genetically identical to the original transformed microorganism. Each genetically identical microorganism is called a clone. The process of producing lots of genetically identical microorganisms is known as cloning. 3. An appropriate host cell for the recombinant DNA : Transformation is the name used to describe the process when a foreign sequence of DNA (such as a gene or cDNA) is introduced into microorganisms such as bacteria and yeast. Two microorganisms that are commonly used in transformations are the bacterium E. coli and the yeast S. cerevisiae. Both microorganisms are single-celled organisms that have fast reproduction rates and thus are quick growing. This makes them ideal for large-scale production in industrial fermenters. E. coli: This is a prokaryote that is often used as a recipient for foreign DNA. Large sequences of foreign DNA can be inserted into E. coli using a plasmid. The DNA is transcribed and translated, and it is possible for the protein coded for by the foreign DNA to account for 60% of the total protein produced by the bacterial cell. E. coli is relatively easy to transform. While there are many advantages of using E. coli, there are some disadvantages – mainly due to the fact that it is a prokaryote and the foreign protein produced may originally have come from a eukaryote. There are some disadvantages of E. coli. The foreign protein produced is not always secreted easily from E. coli. This may be due to E. coli not being able to carry out modifications to the protein after it is made, for example addition of sugar groups. If the protein is not secreted by the bacterium, it causes problems for the biotechnologist as E. coli must be harvested, the bacterial cells broken open (lysed) and the protein purified. This increases the production costs. E. coli does not always fold the foreign protein into its natural three-dimensional shape. This causes the protein to be inactive. UNIT 2: METABOLISM IN MICROORGANISMS (H, BIOLOGY) © Learning and Teaching Scotland 2011 9 METABOLISM IN MICROORGANISMS S. cerevisiae: This is a eukaryote (yeast) that can be used instead of E. coli as the recipient for foreign DNA. Since it is eukaryotic, it can fold proteins into their three-dimensional shape, which allows the proteins to be active. Foreign proteins made by S. cerevisiae are secreted from the cell because S. cerevisiae can carry out post-translational modifications (eg it can add sugar groups to proteins), which allows the proteins to cross the cell wall. Thus proteins secreted by S. cerevisiae can be extracted from the culture medium. Identification of key concepts Recombinant DNA technology allows the transfer of plan t or animal gene sequences to microorganisms to produce plant or animal proteins. Useful genes that that remove inhibitory controls or amplify specific metabolic steps in a pathway can be introduced to increase yield. Restriction endonucleases cut target sequences of DNA , leaving sticky ends. Treatment of vectors with the same restriction endonucl ease forms complementary sticky ends. Ligase combines complementary sticky ends and seals foreign DNA into the plasmid. Suitable microorganisms for transformation include bacteria such as E. coli and yeast such as S. cerevisiae. Identification of particular areas of difficulty Clear visual aids should be used to illustrate the action of endonucleases and the production of sticky ends as students may find the concept difficult to understand. The web link below provides a narrated animation sequence. Links to websites, animations, PowerPoints, audio or video files etc http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations. html# (whole series of biotechnology animations). Co-operative Learning Activities 1 and 5 Other useful information to stimulate interest http://www.sciencedaily.com/news/plants_animals/genetically_modified/ 10 UNIT 2: METABOLISM IN MICROORGANISMS (H, BIOLOGY) © Learning and Teaching Scotland 2011 METABOLISM IN MICROORGANISMS (c) Ethical considerations in the use of microorganisms Links to prior/prerequisite knowledge Students will have already discussed the ethics of stem cell research and sources of stem cells (refer to 2 (ii) Research and therapeutic value of stem cells) and may have carried out the suggested case study related to this outcome. New content areas Consideration of the hazards involved in genetic engineering processes. The policies and practices in place to control risks associated with genetic engineering. Background information The earliest concerns around genetic engineering were that genetic manipulations could create hazardous new pathogens, which might escape from the laboratory. This led to the introductio n of formal guidelines administered by agencies such as the Food and Drug Administration (FDA) in the US and the Medicines and Healthcare products Regulatory Agency (MHRA) in the UK. Today governments throughout the world grapple with how to promote the potential benefits of genetic engineering while ensuring that its products are safe. With new medical products the main cause for concern is the potential for harmful side effects. Hundreds of new genetically engineered vaccines, diagnostic kits and drugs await government approval. Before considered for general marketing, each substance must pass exhaustive tests in laboratory animals and humans. In the case of environmental problems, such as oil spills or chemical wastes that threaten our soil, water and air, genetically engineered organisms may be part of the solution, but their own impact on the environment must be considered before they are widely used. There have been concerns that genetically engineered crop plants could potentially become ‘superweeds’ if they have been engineered to have resistance to herbicides, disease or pests and escape into the wild to overrun native species. Concerns over genetically modified foods have been high profile in the media and the issues raised have included: - the appearance of new allergens - increased antibiotic resistance - the creation of brand new disease-causing organisms, made up of genetic material from many different species. UNIT 2: METABOLISM IN MICROORGANISMS (H, BIOLOGY) © Learning and Teaching Scotland 2011 11 METABOLISM IN MICROORGANISMS Ethical considerations as well as concerns about potential environmental and health hazards will probably slow the application of genetically engineered products. There is always a danger that too much regulation will stifle potential benefits, but the nature of the work clearly requires caution. The challenge seems to lie in striking a safe but productive balance. Identification of key concepts What are the risks associated with genetic engineering? How are these risks managed? What ethical issues have been raised? Identification of particular areas of difficulty While it is important that students are able to form their own opinions on this topic, they must also be able to appreciate the debate as a whole and communicate the issues objectively. Links to sources of further information http://www.beep.ac.uk/content/index.php Links to websites, animations, PowerPoints, audio or video files etc http://www.who.int/foodsafety/publications/biotech/ 20questions/en/index.html http://www.mhra.gov.uk/index.htm http://www.fda.gov/ http://www.geneticallymodifiedfoods.co.uk/ Other useful information to stimulate interest http://www.hse.gov.uk/biosafety/gmo/index.htm http://www.geneticallymodifiedfoods.co.uk/ DVD: Science in Focus, The Virtual Body: Genetic Engineering. Available to buy at www.channel4.com/learning or to view on Teacher’s TV (http://www.teachers.tv/series/science-in-focus-the-virtual-body) Co-operative Learning Activity 2. 12 UNIT 2: METABOLISM IN MICROORGANISMS (H, BIOLOGY) © Learning and Teaching Scotland 2011