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Cloning and Biotechnology

Cloning and Biotechnology

 

Core ideas of each section: 

  • Biotechnology: Use of technology and microorganisms or parts of microorganisms for industrial processes (to meet human needs) 

  • LINKS: genetic engineering 

  • 3 Main Areas: 

    • Cloning

      • Natural Cloning in plants and Animals (important so that we can apply the technique to artificial cloning)

      • Artificial Cloning includes micropropagation in plants and artificial twinning or embryo splitting as well as Somatic Cell Nuclear Transfer in animals 

    • Use of Microbes: 

      • For food production, in medicines and for bioremediation 

      • Microbes in the lab 

        • Sterile technique and growth curves are relevant 

      • Microbes in large scale production

        • Primary and secondary metabolites 

        • Batch and continuous processes  

    • Isolated enzymes and Immobilised enzymes 

 

Cloning:

  • Genetically identical copy of a cell, organism or gene 

  • Genetic uniformity is an advantage when conditions are favourable to allow rapid colonisation of areas where all individuals are equally well adapted to the conditions 

  • In this growth there would be no energy expended in order as no processes such as acting to find partners or gametes 

  • All based on whether there are favourable or changing environmental conditions 

  • Totipotency is important: 

    • Cells that are able to express genes that allow differentiation into ll the ell types needed to reproduce a whole organism (plant cells can be totipotent throughout their life and animal cells are only totipotent in the first few days of fertilisation) 

    • Numerous layers of an embryo and as a result differentiation needs to be allowed for all of  

  • Embryo splitting is needed because of the fact that totipotent cells are only present in animal cells in the first few days of fertilisation 

  • Vegetative propagation: 

    • Bulbs - condensed shoots which is a swollen leaf base with a stem tube 

    • Tubers: Swollen underground stems as found in potatoes 

    • Runners: Do not have food stores and is a lateral stem which grows away from the parent plant and roots develop where the runner touches the ground 

    • Rhizomes: specialised horizontal stem such as marram grass running underground 

  • In a cutting this is an example of horticulture using the process of cutting a part of the stem is cut and grown in a culture and roots will grow. Many crops are grown with cuttings as it preserves desirable characteristics (bananas etc.) 

  • Tissue culture can also be used/micropropagation: 

    • Many ways 

    • Remove some cells from the meristematic tissue of the plant in the [shoot/root tips and lateral buds] - must be meristematic as it is is actively dividing by mitosis and is crucially often virus free, this is important as the crops are genetically uniform so if one part of the crop is diseased then it is likely the whole 

    • This is an explant 

    • This must then be placed in a nutrient medium - the cells will divide by mitosis and form a CALLUS which is an undifferentiated ball of cells. At this point the cytokines can be added to further stimulate mitosis 

    • Callus must then be subdivided, cells are totipotent - each cell is grown on more nutrient medium agar and auxins stimulate differentiation and cell elongation 

    • Roots and shoot develop in a plantlet and can b placed in a compost 

    • NOTE: it is divided just before it divides into root and shoot and is still a callus 

    • This must be done using sterile technique and thus often requires skill staffed and so can add to the cost however it is rapidly developing up 

    • Advatages: rapid, can be used for plants that do not readily produce seeds and may not respond well to vegetative propagation. They may be very rare and be genetically modified and if a plant is required to be pathogen free such as numerous genetically identical crops

    • LEARN ADVANTAGES AND DISADVANTAGES: 

      • A

        • Can be seedless for consumers 

        • Rapid 

        • Culture to produce disease free 

        • Viable number of plats 

        • Provides a method to grow naturally infertile etc. 

        • Increase numbers of rare or endangered 

      • D

        • Monoculture so equally susceptible 

        • Expensive and can require skilled workers 

        • Explants and plantlets are susceptible to disease 

        • Can be inefficient 

        • If the source is infected then all will be affected 

Cloning: Animals

  • Natural cloning in invertebrates are 3 main types (often in diploblastic animals with 2 germ layers as opposed to more complex with 3 like humans) 

    • Budding (yeast and Hydra) 

    • Regeneration (starfish) 

    • Fragmentation (sponges) (almost like glycoproteins for adhesion) 

  • Cloning in vertebrates occurs primarily through the formation of monozygotic twins (identical twins) whereby the early embryo splits to form two separate embryos whereby the fertilisation is totipotent and the early blastocyst splits and thus 2 genetically identical offspring are formed and the same nuclear and mitochondrial DNA 

  • Parthenogenesis: (produce copies without fertilisation) 

 

Artificial: 

  • Artificial Twinning 

    • Begins with natural fertilisation though two organisms with desirable characteristics being chosen and a zygote forming (IVF type method in the lab) 

    • The embryo formed is split into totipotent cells 

    • Each totipotent cell develops into an embryo itself 

    • These embryos are transferred to a surrogate mother 

    • The embryo develops and identical clone offspring is produced 

      • Advantageous as many calf produces 

      • Issue it that they are not clones of the parent they are clones of each other and so there is no guarantee that the desirable characteristics are passed on. As a result farmers may freeze the embryo for a period of time to assure they are suitable and only let 1 grow.

    • In some animals numerous embryos may need to be placed in to a single mother if they naturally produce a littler (pigs) 

    • A: produces many identical copies 

  • Somatic Cell Nuclear transfer 

    • Produces a direct clone of the parent not just clones of the offspring so a sheep with desirable characteristics can be chosen 

    • A somatic cell is removed from a part of the body such as the udder nad cultured for a period of 6 days 

    • An unfertilised eff is also taken from another sheep and the nucleus removed forming a enucleated oocyte (egg cell) 

    • Electrofusion then occurs whereby the egg without a nucleus is fused with a donor cell using a pulse of electricity

    • The embryo will return to a totipotent stage and the embryo resulting from fusion of the udder cell and eff transferred to the uterus of a third sheep which acts as a surrogate mother

    • This surrogate mother gives birth to lamb and so a sheep is genetically identical to the initial, HOWEVER, the mitochondrial DNA will be of the sheep from which the egg was taken so is not an identical clone 

Use all the knowledge of the process to talk about why they are beneficial or useful 

 

Against: 

  • Inefficient as many embryos fail to develop and takes many eggs to produce a single cloned offspring 

  • Many clones have reduced life span (not necessarily the case) 

  • Has been unsuccessful for the reintroduction of almost extinct or extinct animals or rare animals 

 

Already have such a good knowledge and a very good understanding of links so make sure to look through questions and papera as well as legacy papers as well a


 

Use of Microbes: 

  • Advantages:

    • Can be used directly for food production or can be genetically engineered and easily manipulated to form recombinant DNA as they are naked in that they are not associated with a histone protein and have plasmid DNA which enable them to be used as vectors to transfer genes 

    • The plasmids are very easy to extract 

    • No welfare issues to consider 

    • Enormous range of microorganisms capable of carrying out many different chemical syntheses 

    • Low cost as can be grown on waste materials 

    • Can be made to taste like anything 

  • Disadvantages: 

    •  Has little natural flavour 

    • Can produce toxins 

    • Involve Genetic modifications and people do not often want to eat genetically modified things 

    • Needs sterile conditions which adds to cost (could show this with the grown on waste to show both sides) - more akin to high initial set up cost 

    • Protein must be purified to make sure there is no 

 

  • Can be direct use or indirect use

  • Direct is using the microorganisms as a source of food while indirect is using the process to gain a product

  • indirect: Baking and Yeast production. Both require yeast, 

  • Also factors such as yoghurt making, cheese making, make sure to know the microbe, the conditions, why the condition etc. 

  • Direct: Mycoprotein/Corn whereby a single celled fungus is grown in large fermenters using glucose syrup as a food source, can be combined with egg white they are then compressed and formed into a meat substitute

  • Production of Insulin and Penicillin

    • All antibiotics are secondary metabolites and is thus produced during the secondary growth phase  

    • Make sure you are aware of this process etc. 

  • Bioremediation 

    • Use of microorganisms to break down the pollutants and contaminants in the soil or in water 

      • Natural organisms can be used such as nutrients added to oil spills to encourage microbial growth and the oil cja be dispersed into smaller particles to give maximal surface area 

      • GM organisms can be used with scientist developing organisms which can metabolise heavy metals and present it from accumulating in a food chain and risking damage to organisms further up the food chain. The aim would be to remove mercury from contaminated sites 

    • Lab culture 

      • Pure cultures would be of the same colour and note that fungal colonies would be ‘hairier’ as they would have hyphae 

      • Understand aseptic technique well

      • Can do a practical to count colonies and assume that each colony comes from one bacteria and then we work out the number of bacteria in a given vol of the starting 

      • Every serial dilution is a dilution by a factor of 10 if 9 cm3 of water is added 

      • Number of colonies = number of bacteria x dilution of the sample 

    • Growth curve for Bacterial Colonies: 

      • In a closed system there will be a lag phase then exponential growth phase and then a stationary phase and then a death phase. 

      • In the lag phase the genes are beginning to be expressed 

      • If limiting factors are removed then the exponential growth phase will continue 

      • This is key for primary metabolites such as ethanol which is grown assuring that there are no limiting factors that affect the rate of growth through the exponential phase (continuous culture as a result)

      • For secondary metabolites we want the stationary phase (batch culture as a result) 

      • Batch means they are allowed to enter death and then empty and reset with a new group 

      • Continuous they are inoculated into a sterile medium and the culture growth is continually removed and all limiting factor are mitigated to maintain in the exponential growth phase 

      • Limiting factors include: 

        • Nutrients available 

        • Oxygen levels 

        • Temperature 

        • Build up of waste 

      • Note that the products and substrate will be used to increase the biomass of the products 

Food

 

  • Brewing: 

    • Yeast added to a type of grain like barley and other ingredients

    • The yeast respires anaerobically using glucose to produce ethanol via anaerobic respiration. CO2 is also produced 

  • Baking 

    • Bread ingredients are mixed are kneaded to form dough

    • Fermenting the dough for up to 3 hours to enable the yeast respire anaerobically 

    • Cooking the dough which has risen from the dough baked 

  • Cheese: 

    • Bacteria converts lactose to lactic acid in milk and the lactic acid acidifies the milk

    • Acidified milk is then mixed with rennet which contains the enzyme rennin which coagulates the casein protein of the milk in the presence of calcium ions

    • Kappa casein is broken down which stops the casein being kept in solution and so it is precipitated out as a solid held by the calcium ions. This is curd.

    • The curd is then pressed to form cheese

  • Yoghurt: 

    • Fermentation (provides flavour)

    • Bacteria convert lactose to lactic acid which decreases pH causing the milk to coagulate

    • The partial digestion of the milk also aids our digestion of the milk 

    • Other bacteria can be added to aid digestion etc.

  • Penicillin 

    • Fungi produce penicillin to prevent bacteria from growing and competing for resources

    • Grown in industrial fermentors on a large scale 

  • Insulin:

    • Used to treat type 1 diabetics 

    • Can be made by GM bacteria

    • Insulin is produced and collected and then purified and sold as insulin 

  • Bioremediation 

    • Using organisms to remove pollutants like oil from contaminated sites 

    • Bacteria used in oil spills to break down the pollutants into less harmful products 

+ve:

 

  • Faster 

  • Production can be increased/decreased for demand 

  • No ethical issue

  • Good source of protein (mycoprotein)

  • Protein contains no animal fat or cholesterol 

  • Protein content etc. can be edited 

  • Not much land used


 

-ve:

 

  • May not be liked to eat fungi etc.

  • Protein must be purified so it is not contaminated 

  • Infection - setting must be controlled to assure that there are no pathogens etc. 

  • No taste or texture 

 

Isolated Enzymes: Likely to come up 

  • Why use them:

    • We use these as the process is then more efficient and increases yield and reduces the cost of downstream processing

    • Less wasteful as enzymes reused

    • More efficient & increased yield as reused 

    • More specific 

    • Easy to manipulate to different catalytic environment etc.

    • Maximises efficiency as isolated enzymes can be given ideas conditions for animals formulation 

    • Less downstream processing as the product is purer HOWEVER: 

      • Costs are still relatively high due to need to isolate the produce from the enzyme and additionally in the process enzymes may be lost which would increase cost as a greater need to generate and get new enzymes (this is why an enzyme may be attached to a inert support system so when washed over the enzymes are not lost and the product is extracted) 

    • Enzyme not mixed with the product which is beneficial as other enzymes which are mobile are in suspension but the suspension has to be removed which can be costly based on the nature of the enzymes 

  • Negative: 

    • setting up the immobilisation takes time and again can be a bit costly and as they are immobilised the process can be slower

    • Process of immobilising may reduce inefficiency 

    • Higher initial cost of bioreactor 

  • More likely to be extracellular enzymes as they are more robust than intracellular as they can be more robust and able to cope with a variety of temperature and pH 

  • We may still use intracellular if we cannot obtain the required enzyme in extracellular form 

  • Can be immobilised in 3 ways: 

    • Adsorption to inorganic carrier: combination of hydrophobic interactions and ionic links bind them to the supporting surface. These surfaces may be clay, porous carbon, glass bead or resin 

      • The active site could be slightly distorted by the interactions and this can reduce enzyme activity and the bonding forces are not overly strong so the enzymes can become detached. The active site is very accessible and open to the substrate 

      • Adv: cheap an easy an many different processes but enzyme access is easy and very accessible 

      • Dis: Enzymes lost easily 

    • Bonding to an inorganic carrier with ionic or covalent bonds: Can be bonded to a supporting surface such as clay using strong bonds and can be bonded with a cross-linking agent which can link them in a chain 

      • Can be very expensive and the process may distort the active site of the enzyme and reduce activity however they are less likely to detach and leak into a reaction mixture as the bonds are strong 

      • Adv: cost varies and enzymes strongly bonded so less likely to be los

    • Entrapment in a matrix: enzyme molecules trapped in a matrix preventing free movement. Only suitable for processes with a small substrate and product molecule. The matrix can be a cellulose mesh or calcium alginate beads 

      • Disadvantage is that the substrate must diffuse through the whole matrix to gain access to the enzymes in each region and the product has to diffuse out. The benefit is that that there is no way to lose the enzyme molecules as easily 

      • Adv: Widley applicable 

      • Dis: Diffusion of substrate in ad product out 

  • Use: 

    • Glucose Isomerase: role of converting glucose to fructose amd used widely because of many uses and applications of products. Primarily produces high fructose corn syrup which is sweeter than sucrose and is used in diet foods also cheaper than sucrose so used widely 

    • Penicillin acylase (amidase): can be used to form semi-synthetic penicillins such as amoxicillin and ampicillin which can be used as some resistant microorganisms are not resistant to the semi-synthetic forms 

    • Lactase: converts lactose to glucose and galactose by hydrolysis and can be used to produce lactose-free milk. Important to get the calcium etc. from milk so is vital for lactose intolerant people 

    • Aminoacylase: used to produce L-amino acids by removing the acyl group from the nitrogen of an N-acyl-amino acid, L-amino acids are used as building blocks for synthesis of a number of pharmaceutical compounds and can be used as additives for food (for livestock and us) 

    • Glucoamylase: Converts dextrins to glucose from dextrins formed during the hydrolysis of starch and can be used on corn etc. Enzyme used in a wide range of fermentation processes and potentially for an alternative to fuel for motor vehicles 

    • Nitrile hydratase: acts to convert nitriles to amides and can be used for acrylonitrile to acrylamide which can be polymerised and used to thicken plastic. Also for other polyacrylamides to treat water or in paper making

    • Useful in controlling multi-enzyme pathways such that a reaction proceeds in a given order and in sequence to maximise yield

 

Recap: 

 

  • Primary metabolite are produced during normal metabolism this is cultured in continuous culture (such as amino acids, vitamins etc.)

  • Secondary metabolites are produced mainly during the stationary phase of growth so the nutrients are limited to reach the stationary phase faster

  • PENICILLIN IS A SECONDARY METABOLITE 

  • Remember that the growth pattern and graph goes lag phase, log (exponential) phase, stationary phase, death phase 

  • Bioremediation also involves suitable conditions such as available water, suitable temperature, suitable pH etc. 

  • It is beneficial as it uses natural systems, less labour and treatment is insitu. There are also fewer waste products and especially fewer damaging waste products thus less risk of exposure in a clean up. 

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