2.2.1- Outline the differences between reproductive and non-reproductive cloning
Reproductive Cloning: production of offspring which are genetically identical to the mother or the other offspring

Non- Reproductive Cloning: Use of stem cells in order to generate replacement cells, tissues and organs which may be used to treat particular diseases

2.2.2- Describe the production of natural clones in plants using the example of vegetative propagation in elm trees
Vegetative Propagation: The production of structures in an organism that can grow into new individual organisms These offspring contain the same genetic information as the parent so they are clones of the parent.
In Elm Trees: The elm tree is adapted to reproduce asexually following damage to the parent plant.
This happens by separation of some body part of the plant body and its development into a new plant. In an elm tree this occurs by:
Root Suckers/ Basal Sprouts are removed from a tree in autumn and are grown in a nursery bed. They appear within 2 months of destruction.
The suckers grow from meristem tissue in the trunk which is close to the ground (area of least damage)

2.2.3 – Describe the production of artificial clones of plants from tissue culture
Tissue Culture: Large scale cloning
1- A small piece of tissue from the plant is taken to be cloned. It is called an explant
2- In aseptic condition, the explant is placed on a nutrient growth medium which contains sucrose, nutrients and growth hormones
3- Undifferentiated cells in the explant divide via mitosis to produce a callus (Mass of undifferentiated cells)
4- The callus can subdivide many times to increase the number of individual plants at the end
5- Small pieces of callus are transferred onto another growth medium which contains growth hormones
6- This triggers differentiation into shoots and roots
7- The growing plants are taken to a greenhouse where they are acclimatise and grown further before being planted.
2.2.4 – Discuss the advantages and disadvantages of plant cloning in agriculture Advantages | Disadvantages | A lot of genetically identical plants can be produced from one plant | They are genetically identical, so they are all susceptible to pathogens and changes in conditions | The plants can be produced at any time during the year | It requires hard labour – to plant a platelet is harder than to sow a seed | Callus can be genetically engineered | |

2.2.5 – Describe how artificial clones can be produced by nuclear transfer and splitting embryos
Splitting Embryos: Cells from a developing embryo can be separated out to produce a separate genetically identical organism
Nuclear Transfer: A nucleus from a differentiated adult cell is taken and is placed in an enucleated cell. The egg goes through the normal stages of development using genetic information from the nucleus - Dolly the sheep is an example of nuclear transfer.

Advantages | Disadvantages | High value animals can be cloned in large numbers | Ethical objections: Playing God | Rare animals can be cloned to preserve species | Something may go wrong in the process producing practically disabled animals so problems with Animal Welfare | Genetically modified animals can be quickly produced | Cannot cope with changes in environment due to lack of genetic variation | The cloned animal can be genetically tested for disease prior to implantation | Artificially cloned animals may not survive a long time- Dolly died at 6 years |
2.2.6- Discuss the advantages and disadvantages of cloning animals

2.2.7- State that Biotechnology is the industrial use of living organisms to produce food, dugs or other products
Biotechnology: Use of microorganisms or biochemical reactions to produce useful products
2.2.8- Explain why microorganisms are often used in biotechnological processes
Microorganisms:
* Grow rapidly in favourable conditions * Produce chemicals and proteins which can be harvested * Can be genetically engineered to produce useful products * Grow well at low temperatures (cost) * Are not location dependant * Not climate dependant * Generate products in a purer form than chemical processes * Can be grown using nutrient materials which would be toxic to humans
These factors make using microorganisms in biotechnological processes favourable

2.2.9- Describe and explain with the aid of diagrams, the standard growth of microorganisms in a closed culture
Culture: Growth of microorganisms
Lag Phase: Organisms are adjusting to conditions EG. Take in Water Cells are active, no Reproduction so population remains stable.
Log Phase: Population size doubles each generation Population doubles every 20-30 mins.
Stationary Phase: Nutrient levels decrease and waste product concentrations build up Individual organisms die at the same rate at which new individuals are being produced
Decline Phase: Nutrient exhaustion and waste product build up Death rate overtakes reproduction rate

2.2.10- Describe the differences between primary and secondary metabolites

Primary Metabolites: Substances produced by all organisms as part of their normal growth EG. Proteins, Amino Acids
Secondary Metabolites: Substances produced by an organism that is not part of normal growth Only produced by a small number of organisms EG. Antibiotics
2.2.11- Compare and Contrast process of batch and continuous culture

Batch culture: A culture of microorganisms that takes place in a single fermentation. Products are separated from the mixture at the end of fermentation
Continuous Culture: A culture of microorganisms set up in a reaction vessel to which substrates are added and from which products are removed as the fermentation process continues | Batch | Continuous | Growth Rate | Slower- Nutrient levels declines with time | Faster- nutrients are continuously added to the fermenter | Maintenance + Set Up | Easy | Harder | When Contamination occurs… | ….only one batch is lost | ….huge volumes of product is lost | Efficiency | Less – fermenter is not In use all the time | More- Fermenter operates continuously | Useful when… | Producing stuff involving secondary metabolites | Producing stuff involving primary metabolites | Example | Production of Penicillin | Production of Mycoprotein |

2.2.12- Explain the importance of manipulating the growing conditions in a fermentation vessel to maximise product yield
The growing conditions can be manipulate din order to maximise yield Factor | How | pH | Changes in pH in the tank can reduce enzyme activity therefore reducing growth rates | Temperature | Too hot → Denaturation
Too Cold → Slower growth | Type and Addition of Nutrient | Nutrients aid growth
Manipulation depend on primary or secondary metabolite | Oxygen concentration | Most applications use aerobic conditions
Sufficient Oxygen must be available.Lack of Oxygen → Reduction in growth rate |

2.2.13- Explain the importance of asepsis in the manipulation of microorganisms

Asepsis: Absence of Unwanted Microorganisms
Unwanted microorganisms could: * Compete with the culture microorganisms for nutrients and space * Reduce the yield of useful products from culture microorganisms * Cause spoilage of the product * Produce toxic chemicals * Destroy the culture microorganisms and products
2.2.14- Describe aseptic techniques
Aseptic Techniques: Measure taken during a biotechnological process, to ensure contamination from unwanted microorganisms does not occur Techniques for Lab level | Technology for Large scale level | Sterilise equipment before and after use | Washing, disinfecting + steam cleaning the fermenter | Carry out work in a fume cupboard | Make fermenter surfaces from steel to prevent microbes sticking to surfaces | Microorganism cultures are kept closed and away | Sterilising surfaces of all nutrient media to prevent contamination | | Fine filters on inlets and outlets prevent microorganism leaving and entering the vessel |
2.2.15- Describe how enzymes can become immobilised
Immobilised: Any method where the enzyme is held and separated from the reaction mixture, such that the enzyme is not able to freely move
Enzymes that are attached to an insoluble material so they can’t mix with the products
Methods:
Adsorption:
Enzyme molecules are mixed with the immobilising support and bind to it due to a combination of hydrophobic interactions and ionic links.
Covalent Bonding:
Enzyme molecules are covalently bonded to a support using a cross linking agent
Entrapment:
Enzyme may be trapped in a gel bead in their natural state
Substrate and product molecules can pass through the material to the enzyme but the enzyme cannot pass to the solution
Membrane separation:
Separated by a partially permeable membrane.
The substrate and product molecules can pass through the membrane but the solution cannot

2.2.16- Explain why immobilised enzymes are used in large scale production * Enzymes can be recovered easily and then used again many times * Product is not contaminated by the enzyme * Enzyme activity can be controlled more easily * Enzyme is more stable in changing conditions such as temperature * Enzyme is not present with the product so purification costs are low

2.2.17
2.2.18

2.2.19 – Outline how DNA Fragments can be separated using gel electrophoresis
Electrophoresis: Used to separate DNA fragments based on their size
Useful when investigating a crime or preserving species 1. Procedure: 2. DNA Samples are treated with restriction enzyme to cut them into fragments 3. The sample is then placed into a well in one of the gel in an electrophoresis bath which is filled with buffer 4. A direct electric current is passed through the gel 5. DNA fragments are negatively charged due to phosphoryl groups so they travel to the positive electrode 6. Shorter lengths of DNA move faster so move further 7. The position of the fragments can be shown using a DNA Stain

2.2.20- Describe how DNA Probes can be used to identify fragments containing specific sequences
DNA Probe: Short, single-stranded piece of DNA which is complementary to the DNA being investigated
It can be labelled in many ways: * Southern blotting: A nylon/nitrocellulose sheet is placed over the gel, covered in paper towels and left overnight
The DNA fragments are transferred to the sheet and can be analysed * Using a radioactive marker- using the 32P in the phosphoryl groups
Position of the probe can be found by putting an x-ray film over it, so the radioactive emissions can leave black bands * Using a fluorescent marker that emits a colour on exposure to UV light.
The DNA probe can be added to any complementary base pair, via a process called annealing.
This is useful for genetic engineering, genome comparison etc.

2.2.21- Outline how the PCR can be used to make multiple copies of DNA fragments
PCR- artificial DNA replication
Used to make mass copies of DNA samples
PCR works because of these rules:
DNA:
- Made of antiparallel backbone strands
- Made of strands which run from 5’ to 3’
- Grows only from the 3’ end
- Complementary base pairing
PCR can only work with short sections of DNA and requires the addition of a primer in order to start
Primer: Short, single stranded sequences of DNA

The Process: * DNA sample is mixed with DNA nucleotides and DNA polymerase * This mixture is heated to 95®C to break the Hydrogen bonds which hold the base pairs together * Primers are added * Temperature reduced to 55®C, to allow the primers to bind * This forms small sections of double stranded DNA * DNA Polymerase binds to the double stranded section * Temperature is then raised to 72®C which is the optimum temperature of DNA Polymerase * The enzyme then extends the double stranded section by adding free nucleotides to the unwound DNA * When the DNA Polymerase reaches the other end of the DNA Strand, a new double stranded DNA Molecule is generated *