Cloning and biotechnology Flashcards
Natural clones
-clones are genetically identical copies
-term can apply to cells or to whole organisms
-clones are produced by asexual reproduction in which nucleus divides by mitosis
-mitosis creates to identical copies of DNA which are then separated into genetically identical nuclei before cell divides to form two genetically identical cells
-these cells may not be physically or chemical identical as after division they may differentiate to form two different types of cell
-clones are formed in nature
-any organism that reproduces asexually will produce clones of itself
-e.g. single celled yeasts reproduce by budding and bacteria reproduce by binary fission
-both processes involve exact replication of DNA so cells produced genetically identical to each other and parent
Advantages of natural cloning
-if conditions for growth are good for parent they will also be good for offspring
-cloning relatively rapid- population can increase quickly to take advantage of suitable environmental conditions
-reproduction can be carried out even if there is only one parent and sexual reproduction is not possible
Disadvantages of natural cloning
-the offspring become overcrowded
-there will be no genetic diversity (except that caused by mutations in DNA replication)
-population shows little variation
-selection is not possible
-if environment changes to be less suitable whole population is susceptible
Plant cloning by vegetative propagation
-differentiation of many plant cells not as complete as that of animal
-many parts of plant contain cells that retain ability to divide and differentiate into range of types of cell
-this means plants are able to reproduce by cloning
-natural cloning involves process called vegetative propagation
-this is process of reproduction through vegetative parts of plant rather than through specialised reproductive structures
Runners, stolons, rhizomes and suckers
-many plants grow horizontal stems that can form roots at certain points
-these stems are called runners or stolons if they grow on surface of ground and rhizomes if they are underground
-some rhizomes adapted as thickened over wintering organs from which one or more new stems will grow in spring
-suckers are new stems that grow from roots of plant - may be close or further away from base of stem
-in all cases, original horizontal base may die leaving new stem as separate individual
Bulbs
-bulbs e.g. onions, are over wintering mechanism for perennial monocotyldenous plants
-have underground stem from which grow a series of fleshy leaf bases
-also an apical bud which will grow into new plant in spring
-often bulb contains more than one apical bug and each grow to new plant
Corms
-corms often mistake bulbs- are solid rather than fleshy
-is underground stem with scaly leaves and buds
-remain in ground over winter and buds in spring to grow one/ more plants
-croci and gladioli reproduce using corms
Leaves
-Kalanchoe plant reproduces asexually, as clones grow on leaf margins
-immature plants drop off leaf and take root
Tubers
-another type of underground stem
-e.g. potatoes will grow one/more plants
-each new plant then produce many new tubers later that year
Cloning in animals
-animals do not clone as often as plants- very few natural examples
-mammals clone when identical twins form- zygote divides as normal but two daughter cells split and become two separate cells- each cell grows and develop into new individual
-waterfly and greenfly also reproduce asexually
Using nat\ural clones in plants
-for many years gardeners and growers have used vegetative propagation
-easiest way to make clones through cuttings
-to make cutting, stem cut between two leaf joints (nodes)
-cut end of stem then placed in stem- usually from node but may grow from other parts of buried stem
-some plants e.g. geraniums and blackberries will take root easily - others may need further treatment
-dipping cut stem in rooting hormone helps stimulate root growth
-it may also be helpful to wound or remove bark from cut end of stem as this encourages plant to produce callus
-this technique can be used to produce large numbers of plant very quickly
How else can cuttings be made
-root cuttings in which section of root buried just below surface of soil and produces new shoots
-scion cuttings from dormant woody twigs
-leaf cuttings in which leaf placed on moist soil and leaves develop new stems and new roots- some leaves may produce many new plants from one cutting
Tissue culture
-large scale cloning by cuttings can be time consuming and needs a lot of space
-some plants do not respond well to taking cuttings
-many commercially grown houseplants are cloned using tissue culture techniques
-tissue culture is series of techniques used to grow cells, tissues or organs from small sample of cell or tissue
-it is carried out on nutrient medium under sterile conditions
-application of plant growth substances at correct time can encourage cells in growing tissue to differentiate
-tissue culture is widely used commercially to increase number of new plants in micropropagation
Micropropagation
1) suitable plant material selected and cut into small pieces- these often called explants. Explants could be tiny pieces of leaf, stem, root or bud. Meristem tissue often used as this always free from virus infection
2) explants are sterilised using dilute bleach or alcohol. This is essential to kill any bacteria and fungi as these would thrive in conditions supplied to help plant grow
3) explants are placed on sterile growth medium containing suitable nutrients such as glucose, amino acids and phosphates. Gel also contains high concentrations of plant growth substances auxins and cytokinin. This stimulates cells of each explant to divide by mitosis to form callus (mass of undifferentiated, totipotent cells)
4) once callus formed its divided to produce larger number of small clumps of undifferentiated cells
5) these small clumps cells stimulated to grow, divide and differentiate into different plant tissues. This is achieved by moving cells to different growth media. Each medium contains different rations of auxin and cytokinin. First medium contains ratio 100 auxin: 1 cytokinin and this stimulates roots to form. Second medium contains ratio 4 auxin: 1 cytokinin which stimulates shoots form
6) once tiny platelets have been formed, these transformed to a greenhouse to be grown in compost or soil and acclimatised to normal growing conditions
Advantages of artificial cloning
-cloning relatively rapid method of producing new plants compared with growing plants from seed
-cloning can be carried out where sexual reproduction is not possible. Plants that have lost their ability to breed sexually can be reproduced for example commercially grown bananas. Similarly plants that are hard to grow from seed can be reproduced for example orchids in horticulture industry
-plants selected will all be genetically identical to parent plant. Therefore will all display same desirable characteristics such as high yield, resistance to common pest or disease or particular colour of flower
-if original plant had unusual combination of characteristics due to selective breeding or genetic modification then this combination can be retained without risk of losing combination through sexual reproduction
-new plants are all uniform in their phenotype which makes them easier to grow and harvest
-using apical bud as explant for tissue culture ensures new plants are free from viruses
Disadvantages of artificial cloning
-tissue culture labour intensive
-expensive to set up facilities to perform successful tissue culture
-tissue culture can fail due to microbial contamination
-all cloned offspring genetically identical and are therefore susceptible to same pests and/ or diseases. Crops grown in monoculture allow rapid spread of disease or pest between closely planted crop plants
-there is no genetic variation except that introduced by mutation
Artificial clones in animals
-some invertebrate species such as greenfly and waterfleas have evolved ability to clone naturally
-in another species its rare event- therefore most cloning in cells is artificial
-successful cloning starts with cells that are totipotent - such cells can divide and differentiate into all types of cell found in adult organism
-in animals only truly totipotent cells are very early embryo cell
Reproductive cloning
-reproductive cloning can produce large numbers of genetically identical animals
-cloning may be useful for:
-elite farm animals produced by selective breeding (artificial selection) or genetic modification. For example a particularly good individual bull whose value is as stud- supply sperm for artificial insemination
-genetically modified animals developed with unusual characteristics for example goats that produce spider silk in their milk and cows that produce less methane
-two main techniques to achieve reproductive cloning are embryo twinning and somatic cell nuclear transfer
Embryo splitting
-mammals can produce identical offspring if an embryo split very early in development
-this process has given rise to artificial technique
1) zygote (fertilised egg) created by in vitro fertilisation IVF
2) zygote allowed to divide by mitosis to form small balls of cells
3) cells are separated and allowed to continue dividing
4) each small mass of cells placed into uterus of surrogate mother
-this technique has been used to clone elite farm animals or animals for scientific research
-however precise genotype and phenotype of offspring produced will depend upon sperm and egg used- therefore precise phenotypes will be unknown until animal born
Somatic cell nuclear transfer SCNT
-SCNT only way to clone adult
-advantage is that phenotype known before cloning starts
1) egg cell obtained and its nucleus removed, know as enucleation
2) normal body cell (somatic cell) from adult to be cloned is isolated and may have nucleus removed
3) complete adult somatic cell or its nucleus is fused with empty egg cell by applying electric shock
4) shock also triggers eff cell to start developing as though it had just been fertilised
5) cell undergoes mitosis to produce small ball of cells
6) young embryo placed into uterus of surrogate mother
Non reproductive cloning
non reproductive cloning is production of cloned cells and tissues for purposed other than reproduction
Therapeutic cloning
-new tissues and organs can be grown as replacement parts for people who are not well
-skin can be grown in vitro to act as graft over burned areas
-cloned cells have been used to repair damage to spinal cord of a mouse and to restore capability to produce insulin in pancreas
-there is potential to grow whole new organs to replace diseases organs
-tissues grown from patients own cells will be genetically identical and so avoid rejection which is problem when transplanting donated organs
Cloning for scientific research
-cloned genetically identical embryos can be used for scientific research into action of genes that control development and differentiation
-can also be used to grow specific tissue or organs for use in tests on effects of medicinal drugs
Arguments for artificial cloning in animals
+can produce whole herd of animals with high yield or showing unusual combination of characteristics, such as producing silk in their milk
+ produces genetically identical copies of very high value individuals retaining same characteristics
+using genetically identical embryos and tissues for scientific research allows effects of genes and hormones to be assessed with no interference from different genotypes
+testing medicinal drugs on cloned cells and tissues avoids using animals or people for testing
_can produce cells and tissues genetically identical to donor for use in repairing damage caused by disease or accidents
+individuals from endangered species can be cloned to increase numbers
Arguments against artificial cloning in animals
-lack of genetic variation may expose herd to certain diseases or pests
-animals may be produced with little regard for their welfare which may have undesirable side effects such as meat producing chickens that cannot walk
-success rate of adult cell cloning is very poor and method lot more expensive than conventional breeding
-cloned animals may b less healthy and have shorter life spans
-ethical issues regarding how long embryo survive and whether right to create life simply to destroy it
-although endangered species increased population, does not help increase genetic diversity
History of biotechnology
-first coined by Karoly Ereky 1919- used the term to describe any technological process that made use of living organisms or parts of living organisms to manufacture useful products or provide useful services
-this included domestication of animals, planting of crops, mechanisation of agricultural processes and selective processes of plants and animals
-new science of DNA technology brought biotechnology to its current position- out increasing understanding of genetics and genetic engineering along with ability to manipulate living conditions of living organisms has led huge expansion of biotechnology
BIOTECHNOLOGY: food
-ethanol in beer and wine
-carbon dioxide used to make bread rise
-lactic acid used to make cheese and yoghurt
-mycoprotein- filamentous fungus protein used for vegetarian food
-soya- soya beans fermented to produce soy sauce
BIOTECHNOLOGY: pharmaceutical drugs
-penicillin
-other antibiotics
-insulin, other therapeutic human proteins
BIOTECHNOLOGY: enzymes
-protease and lipase used in washing powders
-pectinase used to extract juice from fruit
-sucrase to digest sugar to make food sweeter
-amylase to digest starch into sugar to produce syrup used as sweetener
-protease used to tenderise meat
-lactase to make lactose free milk
-removing sticky residues from recycled paper
BIOTECHNOLOGY: other products
-biogas, combination of carbon dioxide and methane
-citric acid, food preservative
-bioremediation, cleaning waste water
Advantages of microorganisms in biotechnology
-relatively cheap and easy to grow microorganisms
-production processes can take place at lower temperatures saving fuel and reduce costs
-production process can take place at normal atmospheric pressures, safer than chemical reactions which may require very high pressure
-production process not dependant on climate- can take place anywhere with resources to build and run suitable equipment
-microorganisms can be fed by products of other food industries, e.g. starch, waste or molasses, but need to be pre treated
-microorganisms have short life cycle and reproduce quickly- large population can grow very quickly inside fermenter
-microorganisms can be genetically modified easily which allows very specific production processes to be achieved
-fewer ethical considerations to worry about in using microorganisms
-products often released from microorganism into surrounding medium- makes product easy to harvest
Other organisms in biotechnology
-genetically modified mammals e.g. sheep, goats and cows can be used to produce useful proteins
-in some mammals, proteins are incorporated into milk and easily harvested
-e.g. goats have been genetically modified to possess gene for spider silk and secrete it into they milk
-other cases, protein may be secreted into blood
-e.g. GM cows synthesise human antibodies which can be isolated from their blood
Other biotechnology forms
-gene technology
-genetic modification and gene therapy
-selective breeding
-cloning by embryo splitting and micropropagation
-use of enzymes in industrial processes
-immunology
BIOTECHNOLOGY IN FOOD: yoghurt
-yoghurt is milk that has undergone fermentation by Lactobacillus bulgaricus and Streptococcus thermophilus
-bacteria convert lactose to lactic acid
-acidity denatures the milk protein causing it to coagulate
-bacteria partially digest the milk, making it easy to digest
-fermentation also produces flavour characteristic of yoghurt
-other bacteria such as L.casei, L,acidophilus and Bifidobacterium may be added as probiotics- bacteria which may benefit human health by improving digestion of lactose, aiding gastrointestinal function and stimulating the immune system
BIOTECHNOLOGY IN FOOD: cheese
-milk is usually pre treated with culture of bacteria that can produce lactic acid from lactose
-once acidified, milk is mixed with rennet
-rennet contains enzyme rennin (chymosin) found in stomachs of young mammals
-rennin coagulates milk protein (casein) in presence of calcium ions
1) Kappa casein which keeps casein in solution is broken down- this makes casein insoluble
2) casein is precipitated by action of calcium ions which bind molecules together
-resulting solid called curd is separated from liquid component (whey) by cutting. stirring and heating
-bacteria continue to grow producing more lactic acid
-curd is then pressed into moulds
How are the characteristics of cheese determined
-treatment whilst making and pressing curd determines the characteristic of the cheese
-flavour is determined during later ripening and maturation processes
-cheese can be given additional flavour by inoculation with fungi such as Penicilum to produce blue cheese
BIOTECHNOLOGY FOOD: baking
-bread is mixture of flour, water and salt with some yeast (single celled fungus S. cerevisae)
1) mixing- ingredients are mixed together thoroughly by kneading and this produces a dough
2) proving/ fermenting- dough is left in warm place for up to 3 hours whilst yeast respires anaerobically- this produces carbon dioxide bubbles causing dough to rise
3) cooking- risen dough is baked. Any alcohol evaporates during the cooking process
BIOTECHNOLOGY FOOD: alcoholic beverages
-alcoholic beverages are product of anaerobic respiration of yeast
-wine is made using grapes that naturally have yeasts on their skin
-grapes contain the sugars fructose and glucose
-when the grapes are crushed the yeast uses these sugars to produce carbon dioxide and alcohol
-ale or beer is brewed using barley grains beginning to germinate
-as grain germinates it converts stored starch to maltose which is respired by the yeast
-anaerobic respiration again produces carbon dioxide and alcohol
-hopes are used to give bitter taste to liquid
BIOTECHNOLOGY FOOD: single cell protein
-more recently microorganisms used to manufacture protein directly used as food
-fungal protein or mycoprotein is Quorn which was first produced in 1980s
-marketed as meat substitute for vegetarians and a healthy option for non vegetarians as it contains no animal fat or cholesterol
-these fungi can produce protein with similar amino acid profile to animal and plant protein
-they can grow on almost any organic substrate including waste materials such as paper and whey
BIOTECHNOLOGY FOOD: advantages
-production protein faster than that of animal or plant protein
-biomass produced has very high protein content 45-85%
-production can be increased and decreased according to demand
-no animal welfare issues
-microorganisms easily genetic modified to adjust amino acid content
-SCP production could be combined with removal of waste
-production is independent of seasonal variations
-not much land is required
BIOTECHNOLOGY FOOD: disadvantages
-may not want to eat fungal protein or food grown on waste
-isolation of protein- microorganisms need to be isolated in huge fermenters from material they grow on
-protein need to be purified to ensure uncontaminated
-microbial biomass may have high proportion nucleic acids which must be removed
-amino acid profile may be different from traditional animal protein- e.g. deficient in methionine
-infection- conditions needed for microorganisms to grow are also ideal for pathogenic organisms
-palatability- protein does not have taste or texture of traditional protein sources
BIOTECHNOLOGY: scaling up production of drugs
-commercial drug production uses large stainless steel containers called fermenters in which growing conditions can be controlled to ensure best possible yield of product
-temperature- too hot= enzymes denature, too cold= growth limited
-nutrients available= microorganisms require nutrients to grow and synthesise the product. Sources of carbon, nitrogen, minerals and vitamins are needed
-oxygen availability- most microorganisms respire aerobically
-pH- enzyme activity and hence growth and synthesis are affected by extremes of pH
-concentration of product- if product builds up, may affect synthesis
-a fermenter must first be sterilised using superheated steam
-to can then be filled with all components for growth and supplied with a starter culture of microorganism to be used
-culture will be left to grow and synthesise products
Features of fermenter
-pressure vent prevents any gas build up
-air inlet- sterile air provide oxygen in aerobic fermenters
-mixing blades (impellers)
-motor- rotates blades to mix culture evenly
-water jacket inlet- allows circulation of water around fermenter to regulate temperature
-outlet tap for draining fermenter
-inlet for addition of nutrients
-electronic probes to measure oxygen, pH and temperature levels
-air outlets often in ring- air bubbles from outlets mix with culture (known as sparging)
-all inlets and outlets contain filters to prevent contamination
Continuous culture
-some products synthesised by microorganism during normal metabolism when actively growing- called primary metabolites
-such products are continually released from cells and can be extracted continuously from fermenting broth
-broth is topped with nutrients as are used by microorganisms otherwise population becomes too dense
-this is known as continuous culture and keeps microorganism growing at specific growth rate
Batch culture
-other products are produced only when cells are placed under stress such as high population density or limited nutrient availability- these are called secondary metabolites and are produced mostly during stationary phase of growth
-hence culture set up with limited quantity of nutrients and allowed to ferment for a specific time
-after this time fermenter is emptied and product can be extracted from culture- this is known as batch culture
Importance of asepsis
-asepsis is ensuring sterile conditions are maintained
-nutrient medium would also support growth of unwanted microorganisms which would reduce production because unwanted microorganisms:
-complete with cultured microorganisms for nutrients and space
-reduce yield of useful products
-spoil the product
-produce toxic chemicals
-destroy cultured microorganisms and their products
-in processes where food or medicinal chemicals are produced, all products must be discarded if contamination by unwanted organisms occurs
Production of penicillin
-Florey and chain devised process to successfully mass produce penicillin through fermentation by fungus Penicillum chrysogenum
-modern strains of fungus have been selectively bred to be more productive than early strains
-penicillin is secondary metabolite- only produced once population has reached certain size
-therefore penicillin manufactured by batch culture
1) fermenter runs for 6-8 days. Culture then filtered to remove cells
2) Antibiotic precipitated as crystals by addition of potassium compounds. Antibiotic may be modified by action of other microorganisms or by chemical means
3) Antibiotics mixed with inert substances and prepared for administration in tablet form or as syrup or in form suitable for injection
Production of insulin
-insulin widely used to treat type 1 diabetes
-previously extracted from pancreas of animals such as cattle or pigs sent for slaughter
-insulin from slaughtered animals is not identical to human insulin and so less effective than human insulin and expensive to extract
-1978 synthetic human insulin developed by genetically modifying bacterium
-gene for human insulin was combined with plasmid to act as vector so gene could be inserted into bacterium E.coli
-the resulting genetically modified bacterium enabled production of vast quantities of human insulin at relatively low cost
-insulin manufactured by continuous culture
Bioremediation
-bioremediation is use of microorganisms to clean soil and underground water on polluted sites
-organisms convert toxic pollutants to less harmful substances
-involves stimulating growth of suitable microbes that use contaminants as source of food
-requires right conditions for growth of microorganisms: available water, suitable temperature and pH
-where conditions not quite suitable may be modified through addition of suitable substances
-additional nutrients such as molasses may be needed to ensure microorganisms can grow effectively
-may be necessary to pump oxygen in for aerobic bacteria
-where conditions cannot be made suitable in situ, soil may be dug up and moved to be treated ex situ
Advantages of bioremediation
-uses natural systems
-less labour/ equipment required
-treatment in situ
-few waste products
-less risk of exposure to clean up personnel
-HOWEVER bioremediation only suitable for certain products; heavy metals such as cadmium and lead cannot be treated
Growing microorganisms
-microorganisms will grow on any material that provides carbon compounds for respiration and source nitrogen for protein synthesis
-however in lab, microorganisms are grown in growth medium:
-soup like liquid broth, kept in bottles or tubes OR
-set, jelly like substances called agar, melted and poured into Petri dishes
-typical nutrient agar contains peptones (from enzymatic breakdown of gelatine), yeast extract, salts and water
-may also contain glucose or blood
Aseptic technique
-aseptic techniques have been developed to reduce likelihood of contaminating medium with unwanted bacteria or fungi
1) wash your hands
2) disinfect working area
3) have bunsen burner operating nearby to heat air. This causes air to rise and prevent air borne microorganisms settling. Also creates area around it of sterile air in which microbiologist can work
4) As open vessel, pass neck of bottle over flame to prevent bacteria in air entering bottle. Bottle should also be flamed as it is closed
5) do not completely lift lid off Petri dish- just open it enough to allow introduction of desired microorganism
6) any glassware or metal equipment should also be passed through flame before and after contact with desired microorganisms
Techniques in microbiology
-microorganisms can grow almost anywhere
-key is to ensure they grow on medium used and grow desired microorganism rather than others that have infected medium by mistake
-growing microorganisms on agar plates involved 3 main steps
1) sterilisation
2) inoculation
3) incubation
Sterilisation
-nutrient agar medium and any equipment used must be sterilised
-medium sterilised by heating in an autoclave at 121C for 15 minutes (high temperature achieved by boiling water under high pressure inside autoclave)
-this kills all living organisms including any bacterial or fungal spores
-when medium cooled sufficiently to handle it is poured into sterile Petri dishes and let to set
-it is important lid is kept on Petri dish to prevent infection
Inoculation
-inoculation is the introduction of microorganism to the sterile medium- this can be achieved many ways:
-streaking- a wire inoculating loop is used to transfer a drop of liquid medium onto surface of agar. The drop is drawn out into streak by dragging the loop across surface. Take care not to break the surface of agar
-seeding- a sterile pipette can be used to transfer a small drop of liquid medium to the surface of the agar to the Petri dish before the agar is poured in
-spreading- sterile glass spreader may be used to spread the inoculated drop over the surface of agar
-a small cotton swab or cotton bud can be moistened with distilled water and used to collect microorganisms from a surface and then carefully wiped over the surface of agar medium
Incubation
-Petri dish must be labelled and top must be taped to bottom using 2 strips of adhesive tape- be careful not to seal Petri dish completely
-as this can lead to selection of anaerobic bacteria which may be pathogenic
-Petri dish then placed in suitable warm environment such as incubator
-should be placed upside down as this prevents drops of condensation falling onto surface of agar
-also prevents agar medium from drying out too quickly
-suitable temperatures will depend on type of microorganisms being grown
-cultures can be examined after 24-36 hours
-do not open Petri dish
-all petri dishes must be completely sterilised after use and before disposal
-thoroughly wash hands after handling petri dish as any moisture coming out of dish could be source of infection
What may be analysed
-bacteria grow in visible colonies which may be shiny or dull
-some colonies are round with entire edges, while others have crenate edges
-colonies can also be range of different colours
-each colony results from a single bacterium
-filamentous fungi grow into mass of hyphae which may also be circular but mass is not shiny and often looks like cotton wool with fluffy aerial hyphae
-single celled fungi (yeasts) grow as circular colonies
Using liquid medium
-liquid broth initially clear but will turn cloudy when bacteria have grown
-a liquid broth can be useful to increase the numbers of microorganisms before transferring to agar plates for counting or identification
-similar aseptic techniques as described before must be applied when using liquid broth
-liquid broth can be used to identify population growth
Closed culture
-culture which has no exchange of nutrients or gas with external environment
Serial dilution
-sequence of dilutions used to reduce concentrations of a solution or suspension
Population growth in closed culture
-a liquid broth can be used to measure growth rate of microorganism population
-sterile broth inoculated and population size measured at regular intervals of incubation
-population size can be measured by transferring a small sample to an agar plate and incubating agar culture- each individual microorganisms will produce a visible colony
Describe serial dilutions
-its stepwise dilution of broth culture
-at each step, broth diluted by factor of 10
-take 1cm3 sample of broth and add 9cm3 of distilled water- label as 10-1
-take a 1cm3 sample of this diluted broth and add 9cm3 of distilled water- label as 10-2
-continue procedure until series of dilutions with suitable labels
-a drop of dilution can be used to inoculate agar plate
-one of them will produce culture plate in which number of colonies can be counted
-when recording population density, do not forget to multiply count by dilution factor and also by added volume to plate
Importance of serial dilutions
-numbers of individual microorganisms in a broth can be high
-if broth used to inoculate agar plate, may be too many colonies which merge together making counting impossible
-in order to investigate rate of growth of population of microorganisms its essential to reduce population density- achieved by serial dilution
Growth curve
-small population of microorganisms in closed culture that contains all the nutrients required for growth will undergo population growth
-a closed culture refers to population in which conditions set at start and no exchange with external environment
-population growth will follow a predictable pattern
-these are similar to conditions set up for batch production in a fermenter
-in batch production however certain substances such as oxygen may be added to keep population growing until nutrients used up
Describe each stage of growth curve
LAG PHASE
-in early population growth population does not grow quickly because population still small and organisms adjusting to environment
-involves taking up water, cell growth, activating genes, synthesising enzymes
LOG (EXPONENTIAL) PHASE
-in log phase organisms have adjusted to environment
-they each have enzymes needed to survive
-each individual has sufficient nutrients and space to grow rapidly and reproduce
-population doubles in size with each generation
STATIONARY PHASE
-eventually increasing numbers of organisms use up nutrients and produce increasing amount of waste products such as carbon dioxide and other metabolites
-rate of population growth declines and number of individuals dying increases until reproduction rate equals death rate
-this is stationary phase where there is no population growth
DEATH (DECLINE) PHASE
-nutrients run out and concentration of waste products may become lethal
-more individuals die than are produces and population begins to fall
-eventually all organisms will die
Primary metabolites
-primary metabolites produced during normal activities of microorganism will be collected from fermenter during log phase
-in fermenter population not kept in closed culture but conditions are maintained for optimal growth
Secondary metabolites
-secondary metabolites are produced in stationary phase
-population must be kept in closed culture and metabolites can be collected at end of stationary phase or during decline phase
Immobilised enzymes in biotechnology
-some biotechnology processes can be simplified by taking enzymes out of microorganisms
-enzymes are large proteins that act on substrates to generate the product
-enzymes are not used up in the reaction and remain in suspension when reaction has been completed
-in industrial process this means product must be isolated from enzymes before use
-this could be expensive
Advantage of immobilised enzymes being taken out of suspension
-they are held so they do not mix freely with the substrate- means that they also do not mix with the product therefore extraction costs are lower
-continuous processes made easier as there are no cells requiring nutrients, reproducing and releasing waste products
-enzymes are surrounded by immobilising matrix which protects them from extreme conditions- so higher temperatures or wider pH range can be used without causing denaturing
HOWEVER setting up immobilised enzyme process more expensive and immobilised enzymes usually less active than free enzymes making process slower
METHODS IMMOBILISE ENZYMES: adsorption
-enzyme molecules are bound to a supporting surface by a combination of hydrophobic and ionic links
-suitable surfaces include clay, porous carbon, glass beads and resin
-enzyme molecules are bound with active site exposed and accessible to substrate
-however active site may be slightly distorted by additional interactions affecting enzyme activity
-the bonding forces are nit always strong and enzymes can become detached and leak into reaction mixture
METHODS IMMOBILISED ENZYMES: covalent bonding
-enzyme molecules are bonded to a supporting surface such as clay using strong covalent bonds
-enzymes are bonded using cross linking agent, which may also link them in a chain
-the production of covalent bonding can be expensive and distort enzyme active site, reducing activity
-however enzymes are much less likely to become detached and leak into reaction mixture
METHODS IMMOBILISING ENZYMES: entrapment
-enzyme molecules are trapped in matrix that does not allow free movement
-enzyme molecules are unaffected by entrapment and remain fully active
-however, substrate molecules must diffuse into entrapment matrix and product molecules must be able to diffuse out
-method therefore suitable only for processes where substrate and product molecules are relatively small
-calcium alginate beads often used in schools to immobilise enzymes by entrapment whereas industrial use cellulose mesh
METHODS IMMOBILISING ENZYMES: membrane separation
-enzyme molecules separated by reaction mixture by partially permeable membrane
-as in entrapment substrate and product molecules must be small enough to pass through partially permeable membrane by diffusion
-this access to enzymes may limit reaction rate
INDUSTRIAL USE IE: glucose isomerase (xylose isomerase)
-converts glucose to fructose
-probably most widely used enzyme because number of applications of syrup used
-used to produce high fructose corn syrup (HFCS) which is much sweeter than sucrose
-HFCS often used in diet foods as less sugar needs to be added for equivalent sweetness
-may also be used as sweetener in foods for diabetics
-HFCS much cheaper than sucrose and so is widely used in food industry to replace sucrose e.g. in soft drinks, jam, icecream, cereals etc
INDUSTRIAL USE IE: penicillin acylase (penicillin amidase)
-formation semi synthetic penicillins e.g. amoxicillin, ampicillin which were first developed during 1960s
-some microorganisms not resistant to these forms of semi synthetic penicillins
INDUSTRIAL USE IE: lactase
-convert lactose to glucose and galactose by hydrolysis- used to produce lactose free milk
-milk important source of calcium needed for strong bones and teeth
-people with insufficient calcium in diet more likely to develop weak bones or osteoporosis
-therefore important people that are lactose intolerant given calcium supplements or lactose free milk
INDUSTRIAL USE IE: aminoacylase
-a hydrolase used to produce pure samples of L-amino acids by removing acyl group from nitrogen of N-acyl-amino acid
-L-amino acids used as building blocks for synthesis of number of pharmaceutical and agrochemical compounds
-may also be used as additives for human food and animal feedstuffs
INDUSTRIAL USE IE: glucoamylase
-converts dextrins to glucose
-during hydrolysis of starch short polymers of glucose (dextrins) are formed
-hydrolysis by glucoamylase can convert these dextrins to glucose
-glucoamylase can be immobilised on variety of surfaces and used to digest sources of starch such as corn and cassava
-the enzyme used in wide range of fermentation processes including conversion of starch pulp to alcohol used to produce gasohol- an alternative fuel for motor vehicles
-also used within food industry to make high fructose corn syrup
INDUSTRIAL USE IE: nitrile hydratase
-converts nitriles to amides including acrylonitrile to acrylamide
-acrylamide can be polymerised to form polyacrylamide which is a plastic used as a thickener
-most common use of polyacrylamide is treatment of water- it helps to stick many small contaminants together so that they are precipitate or are easy to filter out of water
-polyacrylamide is also used in paper making and to make gel for electrophoresis
