Module 2 Biotechnology and gene technologies 5.2.1- cloning in plants and animals a.

. outline the differences between reproductive and non-reproductive cloning Reproductive cloning Making cloned animals using eggs and sperm. Splitting embryos to make artificial identical twins. Nuclear transfer by taking a differentiated cell from an adult and placing it into an egg with its nucleus. Non-reproductive cloning Using cloned cells to generate cells, tissues and organs to replace those damaged. Use totipotent cells (capable of differentiating into any adult cell). To repair damage, not to make new organisms. Sometime known as therapeutic cloning.

b. Describe the production of natural clones in plants using the example of vegetative propagation in elm trees. Asexual reproduction after damage to the parent plant 1. A healthy elm tree has a root sucker which grows a new tree. 2. The elm tree starts to show signs of dutch elm disease. 3. The main stem is dead but the roots are still alive so root suckers are still produced. 4. Root suckers grow producing new trees. The root suckers are sometimes known as basal sprouts and they grow from the meristem tissue in the trunk close to the ground.

c. Describe the production of artificial clones of plants from tissue culture Taking cuttings o A section of stem is cut between leaf joints (nodes) o The cut end of the stem is treated with plant hormones o The cutting forms a new plant which is a clone of the original parent plant. Grafting o A shoot section of a woody plant is joined to an already growing root and stem (stock) o The graft grows and is genetically identical to the parent plant but the root and stem is not. Micropropgation by callus tissue culture o An explant is taken which is a small piece of tissue from the plant usually from the shoot tip. o The explant is placed on a nutrient growth medium. o A callus which is a mass of undifferentiated cells is formed by cells in the tissue dividing. o After a few weeks, single callus cells are removed from the mass and placed on a growing medium containing plant hormones to encourage shoot growth.

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After another few weeks, the shoots are transferred onto a different growing medium containing different hormone concentrations that encourage root growth. The growing plants (plantlets) are then transferred to a greenhouse to get acclimatised and to grow bigger. They can then be transferred outside

D. Discuss the advantages and disadvantages of plant cloning in agriculture. Advantages Allows new plants to be grown which usually can only be grown from seed e.g. fruit trees. Some plants are sterile so have to be grown by cloning e.g. bananas. It can also allow the growth of crops with useful features e.g. pest and weed resistant plants. The farmer will know that it will be like because of the plant it is grown from. Farmers costs are reduced theyll all be ready to harvest at the same time. Disadvantages They will all be easily susceptible to any new pest, disease or environmental change e.g. the potato famine in Ireland. They are all genetically uniform.

e. describe how artificial clones of animals can be produced. Splitting embryos o Collect eggs and sperm o In vitro fertilisation o In vitro it grows into a 16 cell embryo o The embryo is split into several separate segments o The embryos are implanted into surrogate mothers o Each calf born is a clone

Nuclear transfer o A differentiated diploid cell is taken from an adult e.g. mammary cell from udder in sheep. o An egg (ovum) that is haploid is taken from another adult and the nucleus is removed making an enucleated cell. o Using electro-fusion the nucleus is removed from the differentiated cell and placed into the enucleated ovum. o The cell is then placed into the uterus of another sheep o The early embryo is removed o This embryo is implanted into the surrogate mothers uterus

The sheep is born and is identical to the sheep which the nucleus has come from.

F. discuss the advantages and disadvantages of cloning animals Advantages High value animals can be cloned in large numbers. Rare animals can be cloned to preserve the species. Genetically modified animals can be quickly produced. The transportation of animals for breeding may not be needed. Disadvantages High value animals are not necessarily produced with animal welfare in mind. Genetic uniformity in a species makes it unlikely to cope or adapt to changes. It is still unsure whether animals cloned using nuclear transfer will remain healthy in the long term.

5.2.2- biotechnology a. state that biotechnology is the industrial use of living organism ( or parts of living organism) to produce food, drugs or other products. Biotechnology o Technology based on biology o Involves the exploitation of living organism or biological processes. o Improves agriculture, animal husbandry, food science, medicine and industry

b. explain why microorganisms are often used in biotechnological processes. Production of foods. o Cheese and yogurt making Lactobacillus in milk changes the flavour and texture Bacteria prevent the growth of other bacteria that would cause spoilage o Mycoprotein Quarn/meat alternatives Fusarium is grown in culture Fungal mycelium is produced and separated which is to be processed as a food. o Naturally brewed soya source Roasted soya beans are fermented with yeast or fungi like aspergillus Production of drugs/ pharmaceutical chemicals. o Penicillin Penicillum is grown in culture to produce the antibiotic as a by-product of the metabolism o Insulin

E.coli is genetically modified to carry the human insulin gene. Insulin protein is then secreted as they grow. Production of enzymes/chemical for commercial use. o Pentinase Fruit juice extraction. A. niger grown in certain conditions secretes pectinase enzymes o Calcium citrate Used in detergents A.niger produces citric acid as a by-product of their metabolism o Biogas fuel production Methanogenic bacteria grown on concentrated sewage They respire anaerobically and produce gases that can be used as fuel Biomedication of waste o Waste water treatment Bacteria and fungi use organic waste in the water as nutrients making the water harmless E.g fusarium is grown on corn steep liquor which is a waste product of the dorn milling industry Why use microorganism? o They grow rapidly in favourable conditions, with them being able to double in as little as 30 minutes (generation time) o Can produce protein and chemicals which are released into the surrounding medium where they can be harvested. o Can be genetically engineered to produce specific products e.g insulin. o Can grow well in relatively low temperatures o Can be grown anywhere in the world regardless of climate. o Products tend to be more pure than those generated in chemical processes o Can be grown in nutrient material that would be useless or toxic to humans.

Describe with the aid of diagrams and explain the standard growth curve of a microorganism In a closed culture.

Lag phase o The organisms may be taking in water, undergoing cell expansion, activating specific genes and synthesising enzymes. Log phase o Sometimes known as the exponential phase. o Population size increases each generation o There is enough space and nutrients to reproduce o Low amount of limiting factors o Birth rate is greater then death rate Stationary phase o Nutrients levels decrease o Waste products build up o Birth rate = death rate Decline/death phase o Nutrient exhaustion o Increased level of toxic products. o Birth rate is less then death rate o All organism will eventually die in a closed system.

d. describe how enzymes can be immobilised. Adsorption o Enzyme molecules are mixed with the immobilising support. o The enzyme binds to it because of the hydrophobic interactions and ionic links. o Adsorbing agents: porous carbon, glass beads, clays and resins. o Enzymes can become detached because the bonding forces are not strong (leakages) o But if the enzyme molecules are held so their active site is not changed and is displaced, adsorption can have very high reaction rates. Covalent bonding o Enzymes are covalently bonded to a support by covalently linking enzymes together to an insoluble material like clay. o A cross-bonding link agent is used like gluteraldehyde or sepharose o It does not immobilise a large quantity of enzyme o The binding is very strong so there is very little leakage entrapment o enzymes are trapped in their natural state in a gelbead or a network of cellulose fibres, alginate beads o reaction rates can be reduced because the substrate molecules need to get through the trapping barriers so the active site is easily available. Membrane separation o Enzymes are separated physically from the substrate mixture by a partially permeable membrane o Substrate molecules are small enough to pass through the membrane so the reaction can take place. o Product molecules are then small enough to pass back through the membrane.

e. explain why immobilised enzymes are used in large scale production Enzymes are needed to allow enzyme-substrate complexes to form. The reactions can take place with immobilised enzymes but they do not mix freely with the substrate. Enzymes do not have to be removed so purification costs are lower. The enzymes are immediately available so can be used in continuous processes Immobilised enzymes are more stable because the immobilising matrix protects the enzyme molecules

F. compare and contrast the processes of continuous and batch cultures. Continuous culture Higher growth rate because the process is continuing and nutrients are continuously added. Set up is difficult and maintenance is harder. If contamination occurs, large volumes may be lost. More efficient because the fermenter operates continuously. Useful for primary metabolites. E.g. Human hormones-insulin. Batch culture Growth rate is slower because the nutrient level decreases with time as a fixed amount is added at the start. Easy to set up and maintain. If contamination occurs, only one batch is lost. Less efficient as only one batch is made at a time. Useful for secondary metabolites. E.g. penicillin.

G. describe the differences between primary and secondary metabolites Primary metabolites Substances produced by an organism as part of its normal growth. Includes amino acids, proteins, enzymes, nucleic acids, ethanol, lactate. The production matches the growth Secondary metabolites Substances produced by an organism that are not part of its normal growth. Nearly all antibiotic chemicals produced by a number of microorganisms. The production usually begins after the main growth period and does

in population of the organism. Produced in the log phase.

not match the growth of the organism. Produced in the stationary phase.

h. explain the importance of manipulating the growing conditions in a fermentation vessel in order to maximise the yield. Temperature o If it is too hot the enzymes will be denatured o If it is too cold the growth will be slowed Type and time of addition of nutrient o The growth of microorganism require a nutrient supply including carbon and nitrogen. o Timing can be manipulated depending on whether a secondary or primary metabolite is wanted. Oxygen concentration A lack of oxygen could lead to unwanted products of anaerobic respiration and can reduce the growth rate. PH A change in ph can lead to the reduction of enzyme activity and therefore reduce growth rates.

I the explain the importance of asepsis in the manipulation of microorganisms Asepsis- the absence of unwanted microorganisms. Unwanted microorganism can: o Compete with microorganism for nutrients and space o Reduce the yield of useful products o Cause spoilage of the products o Destroy the culture microorganism and their products.

5.2.3 genomes and gene technologies a. outline the steps involved in sequencing the genome of an organism 1. The genomes are mapped to find out which part of the genome they are from. This can be done by using the location of microsatellites which are short runs of repetitive sequences of 3-4 base pairs. 2. Sample of the genome are mechanically broken to smaller sections of 100,000 base pairs 3. These sections are placed into separate bacterial artificial chromosomes (BACs) and then are transported to e.coli 4. These cells are then grown in culture so many clones of the sections are produced. 5. The cells are then known as clone libraries.

b. outline how gene sequencing allows for genome-wide comparisons between individuals and between species. The identification of genes that code for proteins can be compared Comparing genes of different species can show evolutionary relationship. This is because the more dna sequences that they share, the closer related they are. Comparing genes for the same or similar proteins across a range of organism is known as comparative gene mapping. You can model the effect of changes to genes, for example yeast. By comparing genomes from pathogenic and similar but non-pathogenic organism it can be used to identify the genes or base-pair sequences that are most important in causing a disease which can then lead to developing better drugs and medicines. By analysing the DNA of individuals it can reveal mutant alleles or alleles present that are associated risk of a particular disease.

C. define the term recombinant DNA 1. Recombinant DNA- this is a section of DNA often in a plasmid which is formed by joining DNA from two different sources. D. explains that genetic engineering involves the extraction of genes from one organism and placing them in another. 1. The required gene is obtained. The mRNA is produced from transcription and the gene is synthesised by automated polynucleotide sequencer. A DNA probe is used to locate the DNA fragments. 2. Copy of the gene placed in a vector. It is sealed using DNA ligase and vectors are needed to contain regulatory sequences of DNA 3. The vector carries the gene to the recipient cell. This can be done via: electroporation, microinjection, viral transfer, liposomes. 4. The recipient expresses the gene through protein synthesis. e. Describe how sections of DNA containing a desired gene can be extracted from a donor organism using restriction enzymes. Restriction enzymes cut through DNA at specific points. A particular restriction enzyme will cut where a specific base sequence occurs which is known as the restriction site and it is usually less than 10 base pairs. The enzyme catalyses a hydrolysis reaction to break the sugar phosphate backbones of the DNA double helix Some bases are exposed known as a sticky end.

F. outline how DNA fragments can be separated by size using electrophoresis. 1) DNA samples are cut with restriction enzymes. 2) The DNA samples are placed into wells cut into the negative electrode end of the gel. 3) The gel is immersed into a tank of buffer solution and an electric current is passed through it for a fixed period of time.

4) The DNA is negatively charged so the fragments will diffuse through the gel to the positive electrode end. 5) Shorter lengths will move further in the fixed time 6) A dye is then placed on to show the DNA molecules 7) Via blotting you are lifting the fragments from the gel but to see the DNA you need to label it with a radioactive marker before the samples are run G. describe how DNA probes can be used to identify fragments containing specific sequences The probe is labelled via o A radioactive marker so the location can be seen on exposure to photographic film. o Or a fluorescent marker that emits a colour on exposure to UV light. The probe binds via complementary base pairing to a complementary base sequence which is known as annealing. They can look for specific sequences which is useful to: o Locate a specific gene for genetic engineering. o Identify the same gene on different genomes. o Identify the presence or absence of an allele for a particular genetic disease.

H. outline how the polymerase chain reaction (PCR) can be used to make multiple copies of DNA fragments. 1) The dna is mixed with a supply of DNA nucleotides and DNA polymerase 2) The mixture is heated to 95 Celsius to break the hydrogen bonds making the DNA single stranded. 3) Primers which are short lengths of DNA are added. 4) It is cooled to 55 celcius to allow the primers to bond and form short lengths of double stranded DNA at either end of the sample. 5) The DNA polymerase binds to these double stranded pieces. 6) The temperature is raised to 72 celsius which is the optimum temperature of DNA polymerase. 7) The DNA polymerase adds free nucleotides and when it reaches the other end a new double stranded DNA molecule is generated meaning that there are now two double strands.