22 - Cloning and biotechnology

Natural cloning Vegetative propagation = natural cloning A structure forms which develops into a fully differentiated new plant which is genetically identical to parent Often involves perennating organs which enables the plant to survive adverse conditions They have stored food from photosynthesis and can remain dormant in the soil Bulbs -->  leaf bases swell with stored food and the buds form internally which develop into new shoots and plants when the next growing season arrives Runners --> a lateral stem grows away from the parent plant and roots develop wherever the stem touches the soil, a new plant develops and the runner withers away Rhizomes --> specialised horizontal stem that runs underground often swollen with stored food, buds develop and form new vertical shoots which become independent plants Stem tubers --> tip of underground stem becomes swollen with food or to form a storage organ, buds on storage organ develop into new shoots Using natural cloning in horticulture Splitting up bulbs, removing young plants from runners and cutting up rhizomes are all ways to increase plant numbers cheaply --> new plants genetically identical to parents Take cuttings of a plant --> short sections of stems can be planted directly or in pots Rooting hormone is often applied to the base of a cutting to encourage the growth of new roots Cutting are faster than using seeds and the time from planting to cropping is reduced  It also guarantees the quality of the plant The main drawback is the lack of genetic variation so a new disease or pest could kill them all Cloning sugar cane Internationally important crop to make sugar and biofuels Short lengths with 3 nodes are cut and buried in a clear field of shallow trenches then covered with soil  Per hectare 10-25,000 lengths of stem can be planted

Micropropagation using tissue culture Micropropagation = process of making large numbers of genetically identical offspring from one parent plant using tissue culture techniques This is used when the desirable plant: Doesn't readily produce seeds Doesn't respond well to natural cloning Is very rare Has been genetically modified or selective bred with difficulty Is required to be "pathogen-free" by growers One protocol uses sodium dichloroisocyanurate to keep the plants sterile without being in a sterile lab Basic principles of micropropagation and tissue culture are: Take a small sample of tissue from a plant - the meristem tissue from shoot tips if often dissected out in sterile conditions to prevent contamination The sample is sterilised, usually by being immersed in bleach or ethanol Material removed from the plant is called the explant The explant is placed in a sterile culture medium containing plant hormones which stimulate mitosis The cells divide, forming a mass of identical cells called a callus The callus is divided up and individual cells are transferred into a new culture medium containing a different mixture of plant hormones and nutrients to stimulate development of tiny plantlets The plantlets are potted out to grow and produce a crop Arguments for micropropagation Allows for rapid production large numbers of plants with known genetic make-up Culturing meristem tissue produces disease-free plants Makes it possible to produce viable numbers of plants after genetic modification Way of producing large numbers of plants that are seedless and therefore sterile to consumer tastes Provides a way of producing plants that are usually infertile or difficult to grow from seed Reliable way of increasing numbers of rare plants Arguments against micropropagation Produces a monoculture - susceptible to diseases Relatively expensive and requires skilled workers Explants and plantlets are vulnerable to infection If source material has a virus, all the new plants will too Large numbers of new plants are lost in the process

Natural cloning of animals Common in invertebrates but in vertebrates --> form of twinning Cloning in invertebrates Starfish can regenerate entire organisms from fragments of the original Flatworms and sponges fragment and form new identical animals as part of their normal reproductive process Some female insects can produce offspring without mating (not true clones though, significant differences between mothers and daughters) Cloning in vertebrates Monozygotic twins = identical twins The early embryo splits to form two separate embryos When they're born they may not look identical though - depends on their position in the uterus and the nutrients they received Artificial clones in animals Relatively easy to produce artificial clones of invertebrates - liquidise a sponge or chop a starfish in half Vertebrates are harder, especially mammals Artificial twinning After an egg is fertilised it divides into a ball of cell all of which are totipotent (has the potential to divide into an entire organism) Artificial twinning = the early embryo is manually split and two or more foetuses are developed Used by the farming community to produce the maximum offspring from a particularly good animal A cow with desirable traits is treated with hormones so she super ovulates The ova may be fertilised naturally or by artificial insemination by a bull with desirable traits The early embryos are gently flushed out of the uterus Or the mature eggs are removed and fertilised in the lab Around day 6 the cells are still totipotent so are split into several new embryos  They are then grown in the lab for a few days then inserted into surrogate mothers They develop and are born naturally by different mothers In pigs a number of cloned embryos must be introduced to each mother because they naturally produce a litter Some of the embryos may be frozen Somatic cell nuclear transfer (SCNT) The nucleus is removed from a somatic cell of an adult The nucleus is removed from a mature ovum from a different female animal of the same species (it is enucleated)  The nucleus from the adult somatic cell is placed in the enucleated egg cell and given a small electric shock to make them fuse and begin to divide Sometimes electrofusion is used so the somatic nucleus isn't removed  The embryo that develops is transferred into a third animal where it develops The new animal is a clone of the animal from which the somatic nucleus was taken although the mitochondrial DNA will be from the animal the egg cell was from New animals from this process often suffer from illnesses shown in older animals of their species (Dolly had arthritis at 6) The technique has been improved since Dolly though It is used in pharming --> production of animals that have been genetically engineered to produce therapeutic human proteins in their milk Used to produce GM animals that grow human organs for transplants Arguments for animal cloning Artificial twinning allows high-yielding farm animals to produce more offspring  Artificial twinning enables the success of a male at passing on desirable genes to be determined  SCNT enables GM embryos to be replicated and to develop so you get many from one procedure --> important for pharming SCNT enables scientists to clone specific animals SCNT has potential to enable rare or endangered animals to be reproduced Arguments against animal cloning SCNT is an inefficient process - takes too many eggs to produce a single clone Many cloned offspring fail to develop and miscarry or produce malformed offspring Many animals produced from cloning have shortened lifespans SCNT is relatively unsuccessful so far in increasing populations of rare organisms or allowing extinct species to be brought back to life  

Biotechnology involves applying biological organisms or enzymes to the synthesis, breakdown or transformation of materials in the service of people Most commonly used organisms in biotechnology are fungi, particularly yeasts, and bacteria which are more useful in the newer technologies The use of microorganisms Most biotechnologies involve enzymes in a manufacturing process and the most stable, convenient and effective form of the enzymes is a whole microorganism Used because: No welfare issues to consider - only need optimum conditions for growth Enormous range of them which are capable of carrying out reactions that can be used Can be genetically engineered to carry out reactions they wouldn't naturally Short life cycle and rapid growth rate Nutrient requirements are often cheap and simple All they need is an oxygen supply, relatively low temperatures and food and bring their own enzymes so the processes are kept cheap Indirect food production If the conditions aren't ideal the microorganisms don't grow properly and they don't work as effectively Conditions that are ideal for helpful microorganisms may also be ideal for the food to spoil or pathogens to grow so it has to be sterile Some people have ethical issues with the use of GM organisms Baking Yeast + sugar + water to respire aerobically Carbon dioxide produced makes the bread rise Yeast cells are killed during cooking Brewing Yeast respires anaerobically to make ethanol GM yeasts ferment at lower temperatures to make the process cheaper Cheese-making Bacteria are used They feed on the lactose in milk to change the texture and taste Inhibiting the growth of the bacteria stops the milk going off Yoghurt-making Bacteria are used Often Lactobacillus bulgaricus forms ethanal and Streptococcus thermophilus forms lactic acid Both produce extracellular polymers that give the yoghurt its smooth, thick texture Direct food production Facing protein shortages, scientists have developed ways to use microorganisms to directly produce edible proteins ^^ is single-cell protein (SCP) Most commonly known one is Quorn ^ single-celled fungus that is grown in large fermenters with glucose syrup They are combined with albumen then compressed and formed into meat substitututes High in protein and low in fat Yeasts, algae and bacteria can be used to grow proteins that match animal proteins as well as plant proteins  ^^ can be grown on almost anything, are cheap and low in fat but people have reservations about food grown on waste SCP are used more to feed animals that we prefer to eat like cattle and pigs Advantages of using microorganisms to produce human food Produce proteins faster than plants or animals Have a higher protein content and lower fat content They use a range of waste materials which reduces the cost Can be genetically modified easily Not dependent on weather or mating cycles No welfare issues Can be made to taste like anything Disadvantages of using microorganisms to produce human food Some microorganisms can also produce toxins if the conditions aren't optimum Have to be separated from the nutrient broth and processed to make the food Need sterile conditions which adds to the cost Many people have concerns over eating GM foods The protein has to be purified to make sure its not contaminated People dislike the idea of eating food made on waste Has little natural flavour - have to add additives

Producing penicillin Penicillin chrysogenum needs relatively high oxygen levels and rich nutrient mediums to grow well Is also sensitive to pH and temperature This affects the way its produced commercially  Semi-continuous batch process is used The fungus grows  The fungus produces penicillin Drug is extracted from the medium and purified  Process has relatively small fermenters to maintain the high levels of oxygen The mixture is continually stirred to keep it oxygenated There is a rich nutrient medium The growth medium contains a buffer to maintain a pH of 6.5 The bioreactors are maintained at about 25-27C Making insulin Insulin was originally extracted from the pancreases of pigs or cattle that had been slaughtered for meat Meant the supply was erratic because it depended on the demand for meat Some people were allergic to the animal insulin as it was often impure For some faiths, using pig products wasn't allowed The development of GM bacteria to make human insulin REALLY helped from 1970s onwards The bacteria are grown in a fermener and downstream processing means there's a constant supply of pure human insulin Bioremediation Bioremediation = microorganisms used to break down pollutants and contaminants in soil and water Different approaches: Using natural organisms --> many microorganisms break down organic material to produce carbon dioxide and water Soil and water pollutants are often biological so if these natural organisms are supported they'll break down and neutralise many contaminants For example: in an oil spill, nutrients can be added to encourage microbial growth and the oil can be dispersed into smaller particles to give a higher surface area for microbial action GM organisms --> GM bacteria to break down or accumulate contaminants which they wouldn't naturally encounter Bacteria have been engineered to remove mercury from water --> aim is to develop filters containing these bacteria to remove the mercury In most cases, the natural micoorganisms outperform the GM ones 

Culturing microorganims They need food, the right temperature, oxygen and pH Food = nutrient medium Either in liquid form (broth) or solid form (agar) Enriched nutrient medium allows a small number of organisms to multiply rapidly The nutrient medium must be kept sterile until its ready for use Aseptic techniques are very important Once the agar or broth is prepared, the bacteria has to be added via inoculation Inoculating broth Make a suspension of the bacteria to be grown Mix a known volume with the sterile nutrient broth in the flask Stopper the flask with cotton wool to prevent contamination Incubate at a suitable temperature, shaking regularly to aerate the broth Inoculating agar A wire inoculating loop must be sterilised by being held in a bunsen burner flame until it glows orange Dip the sterilised loop in the bacteria suspension Remove the lid of the petri dish and make a zigzag streak across the surface of the agar Replace the lid of the petri dish and tape it down without sealing it completely so the bacteria have access to oxygen Incubate at a suitable temperature The growth of bacterial colonies They reproduce rapidly --> asexually dividing every minutes in optimum conditions There are four stages to a growth curve: Lag phase --> bacteria are adapting to their new environment --> they're growing and synthesising enzymes bit mot reproducing at their maximum rate Log or Exponential phase --> rate of reproduction close to or at its theoretical maximum Stationary phase --> number of cells formed by binary fission equals the number of cells dying Decline or Death phase --> reproduction has almost ceased and the death rate of cells is increasing Limiting factors that prevent the exponential growth of the bacteria Nutrients available - initially there was plenty of food but as cell numbers increase, the food decreases more rapidly Oxygen levels - as the population rises, so does the demand for oxygen  Temperature - high or low temperatures effect the enzyme-controlled reactions within the cells Build-up of waste - as numbers rise, the build-up of toxic waste does too and can inhibit further growth or be poisonous Change in pH - carbon dioxide produced by the respiration of the cells lowers the pH until it effects enzyme-controlled reactions and inhibits population growth

Primary and secondary metabolites sometimes what is wanted are what's formed as an essential part of the microorganisms' natural function e.g. ethanol, ethanoic acid or range of amino acids aka primary metabolites sometimes the organisms produce substances that aren't required for natural growth but are still used in the cells e.g. pigments, toxic chemicals used for protection aka secondary metabolites Types of bioprocess Batch fermentation microogranisms are inoculated into a fixed volume of medium as growth takes place, nutrients are used up and new biomass and waste build up as the culture reaches the stationary phase, overall growth ceases but they often carry out biochemical changes that form the desired end products process is stopped before the death phase and the products are harvested whole system is cleaned and sterilised before a new batch is introduced Continuous fermentation microorganisms are inoculated into sterile nutrient medium and start to grow sterile nutrient medium is added continuously once it reaches the exponential phase culture broth is continually removed (medium + waste + microorganisms + products) to keep the volume of bioreactor constant enables continuous balanced growth, with levels of nutrients, pH, temperature and metabolic products kept constant both can be adjusted to ensure maximum production of biomass or product --> most are adapted for this majority of industry uses batch or semi-continuous processes continuous fermentation mostly used for the production of single celled proteins and in some waste water treatment useful parts of each mixture has to be separated by downstream processing --> most difficult part of the process Controlling bioreactors Temperature  too low and the mircoorganisms won't grow enough too high and their enzymes will denature bioreactors often have a heating/cooling system attached to temperature sensors to maintain optimum conditions Nutrients and oxygen they can be added in controlled amounts to the broth when probes indicate levels are dropping Mixing things up diffusion isn't enough for the microorganisms to have access to everything they need so bioreactors have big mixing mechanisms to make sure the broth is always circulating  Asepsis if a bioprocess is contaminated it can drastically affect the yield so bioreactors are aspetic units

Isolated enzymes Clear advantages less wasteful --> don't produce excess biomass more efficient --> work at higher concentrations that if whole microorganisms were used more specific --> no unwanted enzymes present maximise efficiency --> can be given ideal conditions for maximum product formation less downstream processing --> pure product is produced Most isolated enzymes are extracellular as they're generally easier and cheaper to use than intracellular  they are secreted so easier to isolate each microorganisms produces few extracellular enzymes so its easy to identify and isolate the desired one they tend to be more robust that intracellular ones (adapted to cope in greater variations of pH and temperature) But intracellular enzymes are sometimes isolated and used because there's a wider range of them and in some cases they're the only ideal one benefits of using very specific intracellular enzymes sometimes outweigh the smaller costs of using an extracellular one glucose oxidase for food preservation asparaginase for cancer treatment penicillin acylase for converting natural penicillin to semi-synthetic drugs Immobilised enzymes isolated enzymes are more efficient that using whole organisms but often very wasteful enzymes aren't cheap and at the end of process are easily lost increasingly, industries are using immobilised enzymes instead (attached to an inert support system which the substrate is passed over and the products are formed) because they are held stationary during the process they can recovered from the reaction mixture and reused --> they don't contaminate the end product so downstream processing is needed less Advantages can be reused so are cheaper easily separated from reactants so less downstream processing - cheaper more reliable because there's a high degree of control over the process they have a greater temperature tolerance as they're less easily denatured easily manipulated so can keep bioreactors running continuously Disadvantages reduced efficiency as the immoblising process may reduce their activity rate higher initial costs of materials as immobilised enzymes are more expensive than free enzymes higher initial costs of the bioreactor as they system is different more technical issues as the systems are more complex How are they immobilised? Adsorption to inorganic carriers (cellulose, silica)  Adv = cheap, simple, used for many processes, enzymes very accessible Disadv = enzymes can be lost from matrix easily Covalent or ionic bonding to inorganic carrier (covalent to carrier with amino group or ionic to polysaccharides) Adv = cost varies, enzymes are strongly bound, very accessible to substrate, pH and substrate concentration have little effect on activity Disadv = cost varies, active site may be modified in the process Entrapment in a matrix (polysaccharides, gelatin) Adv = widely applicable to different processes Disadv = may be expensive, can be difficult, diffusion to and from active site can be slow, effect on activity can vary Membrane entrapment in microcapsules (polymer-based semi-permeable membranes) Adv = relatively simple, relatively small effect on activity, widely applicable Disadv = relatively expensive, diffusion to and from active site can be slow Uses of immobilised enzymes Penicillin acylase --> make semi-synthetic penicillins from natural penicillin to use against penicillin resistant strains or for people who're allergic to penicillin Glucose isomerase --> used to convert glucose to fructose as its sweeter so less needs to be used in foods to make them taste the same  Lactase --> used to make lactose-free dairy products for lactose intolerant people (converts lactose to glucose and galactose) Aminoacylase --> used to produce pure samples of L-amino acids Glucoamylase --> used to complete the breakdown of starch to glucose syrup