Unit 2: Metabolism and Survival Sub-Topic (2.7) Genetic Control of Metabolism (2.8) Ethical considerations in the use of microorganisms Duncanrig Secondary JHM&MHC 2015 Page 1 of 18
On completion of this topic I will be able to: Explain that wild strains of microorganisms can be improved by alteration of the genome (mutation). This could occur naturally, by selective breeding or recombinant DNA technology (genetic engineering). Explain that mutations can be induced by radiation or chemicals. Describe how bacteria can transfer plasmids or pieces of DNA to each other or from their environment. Know that some fungi (eukaryotes) produce variation through sexual reproduction. Explain that plant and animal genes can be transferred to microbes causing them to express the donor gene products (proteins). Know that yields can be increased by gene manipulation. Describe examples of genes which have been introduced to increase yield. Know that safety mechanisms can also be introduced. Describe examples of genes that can be introduced that prevent the survival of microorganisms outside the laboratory setting. Explain that extra chromosomal DNA can be transferred to microorganisms which contain a variety of genes with specific functions. Know that recombinant DNA technology involves the transfer of gene sequences from plants or animals to microorganisms. Describe and explain the structure of commercial plasmids with reference to restriction sites, marker genes, origin of replication, selective markers and regulatory sequences. Describe the process of recombinant DNA technology including the terms restriction endonuclease and ligase. Know that some plant and animal proteins are best expressed in recombinant yeast cells. Describe the ethical issues and explain that biotechnological processes come with some potential hazards and control risks which need to be assessed. Duncanrig Secondary JHM&MHC 2015 Page 2 of 18
Prior Learning Unit 1.1 Cell structure Cells differ in structure as to whether they are animal, plant, fungi or bacterial cells. Nucleus contains genetic information and controls all cell activities. Bacterium has a plasmids, and the chromosomal material is not in a nucleus. Unit 1.2 Producing New Cells Tissue culturing is a technique used to artificially produce new cells. Variables such as oxygen concentration, ph and temperature must be controlled when carrying out tissue culture techniques. Tissue culture techniques must be carried out under sterile conditions by using aseptic techniques. Tissue culture techniques allow tissues to be mass produced. Agar jelly and nutrient broth are both mediums that cells and microorganisms can be grown on. Unit 1.3 Genetic Engineering Genetic information can be transferred from one cell to another naturally or genetic engineering. Bacteria and viruses are used to transfer genetic material from one organism to another. Bacteria have one large chromosome in a ring and smaller rings called plasmids. Genetic engineering is the transfer of DNA from one type of organism to another artificially. The stages in genetic engineering. Genetic engineering is used for the commercial production of medicines such as insulin, human growth hormone and factor VIII. Genetic engineering is used for the commercial production of crops with disease resistance such as potatoes resistant to the Potato Blight virus or tomatoes with a longer shelf life. Duncanrig Secondary JHM&MHC 2015 Page 3 of 18
Genetic Control of Metabolism Some bacteria can naturally transfer plasmids or chromosomal DNA to each other or take up DNA from the environment to produce new strains. Microorganisms are used by humans as they can be induced to make useful products. Wild strains of microorganisms are often cultivated and modified to improve them. They may need improvement because: they lack genetic stability they will not grow on low-cost nutrients they do not produce vast enough quantities of the desired product they are not easy to separate from the product. Therefore strain improvement is carried out to alter the genetic make-up and include addition genetic material to rectify these traits. Techniques used to do this include: mutations & mutagenesis selective breeding and culture recombinant DNA technology 1. Mutations & Mutagenesis Mutations can arise naturally. When a change in the genotype produces a change in the phenotype the organism affected is called a mutant. Mutations are often harmful to an organism. Those which are beneficial are rare. Mutagenesis is the creation of mutations. The frequency of mutations can be increased by exposing the DNA to mutagenic agents. Examples of mutagenic agents include: A wild strain (type) of microorganism is the naturally occurring strain found in nature. A wild strain that may be of use in industry is selected and cultured with optimum growing conditions. Pure strains are then isolated and screened for desired traits. Duncanrig Secondary JHM&MHC 2015 Page 4 of 18
Once the wild strain is isolated it may still be lacking important features. The rate of mutagenesis can be increased by using mutagenic agents, which alter the organisms DNA, resulting in a mutation. If a cell stops making a protein that would have been coded for by a normal gene, it may block the metabolic pathway and intermediate metabolites may build up. If they are desirable substances, then these are harvested for use by humans. Some mutations can be beneficial e.g. a strain that produces higher levels of a desired product. However, mutant strains are often genetically unstable and revert to the wild type in continuous culture. Therefore, it is very important that an improved strain of a microorganism is carefully monitored to make sure that it is the mutant that is selected for use. 2. Selective Breeding and Culture Selective breeding is the process by which humans deliberately cross different strains of organisms to produce new strains showing the desirable characteristics from each parent. Duncanrig Secondary JHM&MHC 2015 Page 5 of 18
Genetic Transfer Scientists also attempt to produce new strains of useful bacteria by creating conditions where transfer of genetic material may occur. New strains of bacteria can occur by horizontal transfer of genetic material this occurs naturally when: Bacteria transfer plasmids or pieces of chromosomal DNA from one strain to another. Bacteria take up and incorporate fragments of DNA from their environment. This uptake, incorporation and expression of DNA from a bacterium's surroundings is called transformation. Horizontal Transfer Use Torrance Fig 13.6 to label the following diagrams, and beside them, write a description of the process. Duncanrig Secondary JHM&MHC 2015 Page 6 of 18
Transformation In some species transformation occurs naturally when the cell takes up a piece of foreign DNA from the remains of a cell that has been destroyed and has undergone lysis. Duncanrig Secondary JHM&MHC 2015 Page 7 of 18
3. Recombinant DNA technology Scientists can use recombinant DNA technology to reprogramme microorganisms. This means scientists can transfer plant or animal gene sequences to microorganisms to make them produce plant or animal proteins. Scientists can also use this technology to introduce a gene or genes to existing microorganisms to: Improve yield by amplifying a specific metabolic step or removing an inhibitory control that affects it Improve extraction of product by causing a cell to secrete the product into surrounding environment Add a safety mechanism - make the microorganism incapable of surviving in another environment A gene from one organism (e.g. a human or a plant) is transferred into a vector (plasmid or artificial chromosome) of a microorganism so that the microbe produces the protein that the gene codes for. Duncanrig Secondary JHM&MHC 2015 Page 8 of 18
The previous diagram shows the introduction of a gene into a bacterium via a plasmid. The protein produced by the microorganism could be Penicillin Recombinant DNA Technology is a highly skilled process with many factors which need to be taken into account and carefully controlled, including the following: Enzymes Two types of enzymes are required in recombinant DNA technology one type to cut open chromosomes and the other to seal pieces of DNA together. 1. Restriction endonucleases These are enzymes which recognise specific short sequences of bases on DNA (about 4 8 nucleotides in length), known as restriction sites. The sequence can be found on both of the DNA strands and may be repeated at intervals along its length. The restriction enzyme causes a cut to be made every time it meets this sequence. The cut can either produce blunt ends or sticky ends. If the cut goes straight through the DNA molecule, blunt ends will be produced as the two strands of nucleotides will be cut at the same place. Duncanrig Secondary JHM&MHC 2015 Page 9 of 18
If the nucleotides are cut at different points (several nucleotides apart), each strand will be left with a short single stranded sequence sticking out. As restriction enzymes always cut at their own specifically recognised sequences, the same enzyme must be used to cut out the desired gene and o open the plasmid into which it is to be inserted. This ensures complementary sticky ends. 2. DNA ligase DNA ligase is an enzyme which seals ends of DNA fragments together by making bonds form between them. It is therefore applied to seal the introduced gene into the plasmid before it is inserted back into a bacterium. Ligase enzyme seals DNA at these points Duncanrig Secondary JHM&MHC 2015 Page 10 of 18
Vectors A vector is something that can have DNA from another source inserted into it. Recombinant (bacterial) plasmids Artificial chromosomes are used as vectors to carry DNA from one organism (e.g. human gene) into another (e.g. bacteria). This brings about transformation in the bacterial cell. 1. Plasmids as vectors To be effective as a vector, a plasmid must have the following features: a. Restriction Site that is cut by the same restriction endonuclease that cut the gene to be transferred in. This ensures the sticky ends are complementary to the gene being inserted. Duncanrig Secondary JHM&MHC 2015 Page 11 of 18
b. Marker Gene to allow the scientist to determine whether the host cell has taken up the plasmid vector, e.g. if the plasmid takes up a gene giving resistance to an antibiotic, the cells are then cultured in a medium containing that antibiotic. Any cells that failed to take up the recombinant plasmid will be killed by the antibiotic, leaving only the ones with the modification. Fluorescent proteins can also be used as markers. Bacteria with the gene inserted do not turn blue c. Origin of Replication these genes control self-replication and regulatory sequences that allow control of existing genes and the expression of the inserted gene. This is essential if the transformed cell is to make more copies of the plasmid and therefore make more of the desired product. Use Torrance Fig 13.10 to label these three sites on the plasmid. Duncanrig Secondary JHM&MHC 2015 Page 12 of 18
To be an effective vector the plasmid must have: a. b. c. 2. Artificial Chromosomes as Vectors Artificial Chromosomes have been developed by Scientists to act as vectors. They have all 3 features described for plasmids (restriction site, marker gene and origin of replication). However, in addition to this they are also able to carry much more foreign DNA than a plasmid. Why might a DNA technologist choose to use an artificial chromosome as a vector instead of a plasmid? Duncanrig Secondary JHM&MHC 2015 Page 13 of 18
Strain improvement New genes introduced to a microorganism, such as a bacterium or a yeast cell can confer an advantage on that organism by; Improving the steps in a metabolic pathway or removing inhibitory parts of it in order to improve the yield of production of a substance Causing the cell to secrete the product allowing it to be harvested more easily Acting a safety mechanism by ensuring that the altered microorganism cannot survive in the wild, removing the possibility of it breeding with wild strains. Limitations The DNA of eukaryotes contains protein coding sections called as well as non-coding DNA known as. The DNA of prokaryotes, such as bacteria, has exons but no introns. When DNA is transcribed into mrna in a eukaryote, the primary transcripts are modified by splicing to remove introns (see previous notes on gene expression). In addition to this, once the protein has been produced it is often modified further (e.g. folded in a particular way or another substance added to it). Neither of these processes occurs in prokaryotes. As a result of these differences, problems can arise when a gene from a eukaryote is expressed by a prokaryote. It may be that the protein produced does not function at all as it is not folded properly or that it breaks down before it can be collected. In order to overcome these difficulties, it is sometimes necessary to use genetically modified eukaryotic cells, such as yeast, to avoid polypeptides being folded incorrectly or lacking some other post translational modification. Duncanrig Secondary JHM&MHC 2015 Page 14 of 18
Answer the Testing Your Knowledge questions from Torrance Page 216. Ethics Describe the ethical considerations under both headings below: Profit Patents Duncanrig Secondary JHM&MHC 2015 Page 15 of 18
Hazards and Risks How long does it take to introduce a new microbiological product onto the consumer market? Before the new product is granted a licence to manufacture and sell, what must they ensure? Complete the risk assessment cycle below: Research task: Duncanrig Secondary JHM&MHC 2015 Page 16 of 18
Research the development of a microbiological product from discovery to market. Name the product and explain its use Describe each step of the process from development to market Summarise how long the process took, the problems encountered and the benefits achieved from its availability Duncanrig Secondary JHM&MHC 2015 Page 17 of 18
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