cloning and biotechnology

uses for microorganisms

• food

• medicine

• bioremediation

food as a use of microorganisms :

indirect :

• baking

• brewing

• cheese

• yoghurt

direct :

• quorn

medicines as a use for microorganisms :

• penicillin

• insulin

bioremediation as a use of microorganisms :

• natural processes

• genetically modified organisms

brewing :

• yeast respires anaerobically (fermentation)

• called Saccharomyces cerevisiae

• glucose (sugar) = ethanol + carbon dioxide

• C6H12O6 = 2C2H5OH + 2CO2

• malting

• mashing

• fermentation

• maturation

• finishing

malting during brewing :

• barley germinates

• enzymes break down starch into sugars which yeast can then respire

mashing during brewing :

• malt mixed with hot water (55-65C’)

• enzymes break down starch producing wort

fermentation during brewing :

• yeast added to wort

• fermentation occurs

• yeast eventually killed as pH lowers

• ethanol builds up

• oxygen decreases

maturation during brewing :

• beer conditioned for 4 to 29 days

• at 2-6 degrees

finishing during brewing :

• beer filtered

• pasteurised

• and packaged

baking :

• uses yeast (Saccharomyces cerevisiae) which respires aerobically

• carbon dioxide is produced causing the bread to rise

• glucose + oxygen = carbon dioxide + water

process of baking :

• active yeast mixture added to flour and other ingredients

• left in warm environment

• excess air removed from dough

• dough kneaded, shaped and allowed to rise again

• cooked in hot oven

• CO2 bubbles expand

• yeast cells killed during cooking

cheese making :

• bacteria converts the sugar lactose in milk into lactic acid

• uses lactococci and lactobacilli etc..

process of cheese making :

• milk is pasteurised at 95 degrees for 20 secs

• kills off natural bacteria

• milk is homogenised to evenly distribute fat droplets

• bacteria and chymosin enzymes are added to help clot the milk

• milk separates into solid curls and liquid whey

• cottage cheese - curds packaged and sold

• harder cheese - curds cooked in whey and strained using a cheese cloth to be packaged or matured

• whey - used to feed animals

yoghurt production :

• bacteria feed on milk sugar called lactose to produce lactic acid

• uses the bacteria Streptococcus thermophilus and Lactobacillus bulgaricus

process of yoghurt production :

• equipment sterilised - heated at 71 degrees for 20 secs

• milk is pasteurised

• bacteria culture is added and incubated

• yoghurt sampled

• flavour, colour is added and packaged

• quality control occurs

• some culture is taken to add to the next batch

what is biotechnology?

applying biological organisms or enzymes to the synthesis, breakdown or transformation of materials in the service of people

advantages of using microorganisms

• no welfare issues

• huge range of suitable microorganisms

• genetic engineering allows the manipulation of microorganisms to produce otherwise unlikely products e.g. human insulin

• short life cycle

• rapid growth rate

• nutrient requirements very minimal and cheap

indirect food production :

• used in biotechnological processes to produce food

• microorganisms have an important impact on other food

• baking

• brewing

• cheese

• yoghurt

direct food production :

• e.g. quorn - a fungus

• single cell protein

• grown in large fermenters using glucose syrup as a food source

advantages of using microorganisms to produce human food :

• reproduce and produce protein quickly

• high protein content with little fat

• uses waste products from humans and animals reducing cost

• can be genetically modified

• flavours can be added

• not dependent on weather

• no welfare issues

disadvantages of using microorganisms to produce human food :

• some may produce toxins

• must be separated from nutrient broth to make food

• needs carefully controlled sterile conditions

• concerns with genetic modification

• protein must be purified

• lacks flavour

penicillin :

• first effective antibiotic

• produced by the mould Penicillium notatum

• later penicillium chrysgenum used to produce a bigger yield

• needs high oxygen levels and rich nutrients

• pH and temperature must also be monitored

process of producing penicillin :

• uses small small fermenters to maintain high levels of oxygenation

• mixture continuously stirred

• rich nutrient medium present

• growth medium contains a buffer to maintain pH of 6.5

• bioreactors maintained 25-27*C

industrial uses of immobilised enzymes :

• glucose isomerase

• penicillin acylase

• lactase

• aminoacylase

• glucoamylase

primary metabolites :

• essential compounds used in the metabolic activities of an organism

• used for growth, development and reproduction

• e.g. ethanol, ethanoic acid, amino acids and enzymes

secondary metabolites :

• substances produced by organisms which arent essential for normal growth but are still used in cells

• e.g. pigments, chemical defense systems of plants

• often required in a bioprocess

• organism will not suffer short term without these strategies

bioprocesses :

• once an microorganism has been chosen as well as the size and shape of bioreactor, the organisation of the commercial production must be decided

• batch fermentation

• continuous fermentation

batch fermentation :

• microorganisms are inoculated into a fixed volume of medium

• growth takes place using up nutrients

• new biomass and waste products build up

• overall growth decreases as culture reaches stationary phase

• microorganisms carry out biochemical changes to form the end products

• e.g. antibiotics and enzymes

• process stopped before end phase and end products collected

• system then cleaned and sterilised for new starter batch of culture

continuous culture :

• microorganisms inoculated into sterile nutrient medium

• sterile nutrient medium constantly added to culture once exponential point of growth reached

• culture broth continuously removed

• the culture volume in the bioreactor constantly the same

• allows for continuous balanced growth where levels of nutrients, pH and metabolic products kept constant

bioreactors :

• bioreactors can be adjusted to maximise production of biomass or metabolites

• most systems adapted for maximum yield

• produce mixture of unused nutrient broth, microorganisms, primary metabolites and waste products

• useful part separated through downstream processing

factors needing control in bioreactors :

• temperatures

• nutrients and oxygen

• asepsis

• mixing

temperature of bioreactors :

• too low temp - microorganisms will not grow quickly enough

• too high temp - enzymes denature and microorganisms destroyed

• therefore heating/cooling system operates a negative feedback loop to maintain optimum conditions

nutrients and oxygen in bioreactors :

• oxygen and nutrient medium added to reactor in controlled amounts

• probes and sample tests indicate the levels of these

mixing in bioreactors :

• diffusion isnt enough to supply all microorganisms with enough oxygen and nutrients

• therefore mixing system is necessary to maintain stable, continuous conditions throughout the bioreactor

asepsis :

• contamination from other microorganisms can affect yield

• therefore most bioreactors are sealed aseptic units

advantages of isolated enzymes :

• less wasteful - whole microorganisms use up substrate which produces biomass rather than product

• more efficient - isolated enzymes work at higher concentrations

• more specific - no wasteful side reactions take place

• maximises efficiency - isolated enzymes can be given their specific optimum conditions which could be different to that of the whole organisms

• less downstream processing - isolated enzymes produce pure product, whole microorganisms produce variety of products therefore more expensive

advantages of producing extracellular enzymes using isolated enzymes :

• secreted therefore easy to isolate

• microorganisms produce few therefore easy to identify

• microorganisms produce hundreds of intracellular enzymes which would then have to be extracted

• extracellular more robust - able to adapt to harsher temps and pH

why might intracellular enzymes still be used in biotechnological processes :

• there is a bigger range of intracellular enzymes, therefore may be ideal in some cases

• e.g. glucose oxidase in food preservation

• e.g. penicillin acylase for converting natural penicillin into semi synthetic drugs

using immobilised enzymes :

• immobilised enzymes attached to inert support system

advantages of using immobilised enzymes :

• can be reused therefore cheaper

• easily separated from reactants and products reducing downstream processing

• more reliable due to higher degree of control

• greater temperature tolerance - less easily denatured by high temperatures

• easier to manipulate - the catalytic properties can be altered to fit a particular process

disadvantages of using immobilised enzymes :

• reduced efficiency - immobilising an enzymes reduces its activity rate

• higher initial cost

• higher cost of bioreactor

• more technical issues - reactors are more complex and therefore more expensive to replace when broken

surface immobilisation (adsorption) :

• adsorption to inorganic carriers

• e.g. cellulose, silica

• advantages - simple and cheap, can be used with many processes, enzymes very accessible to substrate

• disadvantages - enzymes can be lost from the matrix easily

surface immobilisation (covalent/ionic bonds) :

• covalent or ionic bonding to inorganic carriers

• advantages - enzymes strongly bound and unlikely to be lost, enzymes accessible to substrate, ph and substrate concentration have little effect

• disadvantages - cost varies, active site might be modified making it less effective

entrapment :

• occurs in the matrix

• e.g. polysaccarides, gelatin, activated carbon

• advantages - widely applicable

• disadvantages - expensive, difficult to entrap

membrane entrapment :

• membrane entrapment in microcapsules or behind semi permeable membrane

• advantages - simple, small effect on enzyme activity, widely applicable

• disadvantages - expensive, diffusion of substrate to and from the active site is slow

immobilised penicillin amylase :

• used to make semi synthetic penicillins from naturally produced penicillin

• made to combat penicillin resistance

• important for treating bacteria which is resistant to the original penicillin

immobilised glucose isomerase :

• used to produce fructose from glucose

• due to fructose being much sweeter than sucrose or glucose as used in the food sweetener industry

• glucose is produced cheaply

• glucose isomerise is then used to turn cheap glucose into fructose

immobilised lactase :

• used to produce lactose free milk

• immobilised lactase hydrolyses lactose to glucose and galactose

immobilised aminoacyclase :

• used to produce pure samples of L amino acids

• used in the production of pharmaceuticals, organic chemicals, cosmetics, food

immobilised glucoamylase :

• used to complete the breakdown of starch to glucose syrup

• amylase enzymes break starch down into short chain polymers called dextrins

• the breakdown of dextrins to glucose is catalysed by immobilised glucoamylase

immobilised nitrile hydratase :

• enzymes which is used in production of plastic

• immobilised nitrile hydratase is used to hydrate acrylonitrile into acrylamide

• this important compound is then used in the plastics industry

natural cloning in plants :

• form of asexual reproduction

• occurs in many species of flowering plants

• form fully differentiated new pant which is genetically identical to parent

• often involves perennating organs which enable plants to survive in harsh conditions as they store food

examples of natural cloning :

• bulbs - leaf base swells with stored food in which a bud forms eventually

• runners - e.g. spider plant

• rhizomes - e.g. marram grass, specialised stem underground swollen with stored food which grow buds

• tubers - stored food which becomes a bud

use of natural clones in horticulture :

• used by farmers to produce new plants

• propagation also used - either dipped in rooting hormone or directly in the ground

• much faster than growing from seeds

• also guarantees quality of the plant as identical to parent

• however will lack genetic variation therefore susceptible to diseases

example of natural cloning in horticulture :

• sugar cane

• internationally used to make sugar and manufacture biofuels

• fastest growing crop

• propagated by burying 30cm of sugar cane with 3 nodes covered in a thin layer of soil

artificial cloning in plants :

uses the many totipotent plant cells to create many identical clones

micropropagation using tissue culture - artificial cloning :

• uses tissue culture to create many identical offspring from one parent plant

• one technique uses sodium dichloroisocyanurate which maintains sterile conditions

• ensures safety of endangered plants

why use micropropagation to clone plants?

• when a plant doesn’t easily produce seeds

• plant doesn’t reposed well to natural cloning

• rare or endangered species

• species hard to breed due to GM or selective breeding

• is required to be pathogen free for growing food

process of micropropagation and tissue culture :

• small sample of meristem tissue taken in sterile conditions

• sample is sterilised using bleach/ethanol/ sodium dichloroisocyanurate

• explant paced in sterile culture medium containing balanced plant hormones

• these stimulate mitosis forming a callus

• the callus is divided and transferred into separate culture medium containing different plant hormones

• this causes growth pf plantlets

• eventually potted into compost and then outside

advantages of micropropagation :

• allows for rapid yield and ensures good crops

• culturing meristem produces disease free plants

• allows GM plants to breed

• can produce sterile and seedless crops ready for consumption

• can prevent extinction of rare plants

disadvantages of micropropagation :

• produces monoculture therefore susceptible to disease

• expensive and requires skilled workers

• the explants and plantlets are very vulnerable to infection

• if sterile conditions aren’t maintained all the clones will be infected

natural animal cloning :

• common in invertebrates not vertebrates

• occurs in the form of twinning

natural cloning invertebrates :

• e.g. starfish regenerate entire animals from fragments of the original when damaged

• e.g. flatworms normal reproductive process involves cloning the original

• e.g. hydra produce buds on the side of their body which grow into identical clones

• e.g. some female insects can reproduce without a male

• the difference between mother and daughters may be due to high mutation rates not genetic variation

natural cloning in vertebrates :

• the formation of identical twins

• at an early stage the embryo splits into 2

• producing genetically identical offspring

artificial cloning in animals :

• easy to clone starfish or sponge as they will regenerate themselves

• two techniques are widely used :

  • artificial twinning

  • somatic cell nuclear transfer

artificial twinning :

• in the early stages of development of an embryo the cells are totipotent

• in artificial twinning the embryo is split manually into multiple pieces

• used in farming industry to produce maximum offspring from good milk producers

• produces multiplier genetically identical offspring

process of artificial twinning :

• a cow with desirable traits is treated with hormones to increase ovulation

• the ova is fertilised by a bull with desirable traits

• the early embryo is flushed out of the uterus

• IVF could also be used instead outside of the cow

• after 6 days the cells are still totipotent and are split into multiple smaller embryos

• the split embryos are grown further in a lab and then implanted into surrogate mother

• embryos then develop as usual;l in the uterus and are born naturally

somatic cell nuclear transfer :

• dolly the sheep was the first successful cloned adult animal

• involves transferring the nucleus into an enucleated egg cell which is then implanted into a surrogate mother

• animals of different breeds are used to easier identify the original animal in which the nucleus was used from

• used in pharming and producing GM animals which can be used for organ transplants

what is pharming :

the production of animals which have been genetically engineered to produce therapeutic human proteins in their milk

process of somatic cell nuclear transfer :

• nucleus removed from a somatic cell of an adult animal

• the nucleus of a mature ovum is removed from a different female of the same species

• the nucleus of the somatic cell is placed into the enucleated egg cell

• a mild electric shock fuses them together allowing it to divide

• the embryo is then transferred into the uterus of another animal and is given birth naturally

• the clone is of the animal from which the somatic cell nucleus was used

• the mitochondrial DNA would have come from the egg cell

advantages of animals cloning :

• enable high yield farm animals to produce more desirable offspring

• enable succession of desirable genes of the male animals

• allows GM embryos to be replicated and develop to produce many from juts one GM embryo

• enables scientists to clone specific animals - popular dogs and cats

• allows rare animals too reproduce

disadvantages of animal cloning :

• inefficient process - many different eggs are needed from different animals

• many fail and have miscarriages or produce malformed

• cloned animals have shortened life spans

producing penicillin :

• needs high oxygen levels and a rich nutrient medium

• also affected by temp and pH

• uses semi continuous batch process

• the first stage allows the fungus to grow

• the second stage the bacteria produce penicillin

• the last stage the drug is extracted and purified

process of producing penicillin :

• uses small fermenters due to it being harder to maintain high oxygen levels in large bioreactors

• mixture continuously stirred to keep oxygenated

• surrounded by rich nutrient medium

• contains buffer to maintain pH at 6.5

• temperature maintained at 25-27 degrees

bioremediation :

• using microorganisms to break down pollutants and contaminate in the soil/water

• can use two different approaches

  • using natural organisms

  • using GM organisms

using natural organisms for bioremediation :

• many microorganisms break down organic material to produce CO2 and water

• therefore can break down and neutralise many contaminants like sewage and crude oil

• nutrients can be added to encourage microbial growth

• contaminate can also be dispersed for larger surface area for microbial action

using GM organism for bioremediation :

• trying to develop GM bacteria to break down contaminates which don’t naturally occur

• e.g. creating filters containing bacteria which can filter out the contaminate

risks when culturing microorganisms :

• risk of mutations creating a pathogenic strain

• contamination of pathogenic microorganisms form the environment

culturing microorganisms :

• they require food, and the right conditions of temperature, pH, and oxygen

• nutrient medium also required to increase microbial growth

• aseptic techniques also must be used to prevent contamination

• once the agar/nutrient broth is prepared the bacteria have to be inoculated

inoculating broth :

• create a suspension of bacteria

• mix a known volume with a sterile nutrient broth in a flask

• stopper flask with cotton wool to prevent contamination

• incubate a suitable temperature

• shake regularly to aerate to provide oxygen for bacterial growth

inoculating agar :

• sterilise wire inoculating loop using a bunsen burner

• allow to cool without touching any surfaces

• dip sterilised loop in bacterial suspension

• remove lid and make a zig zag streak across the agar

• replace lid of petri dish

• close but not seal to allow oxygen in

• incubate at suitable temperature

the growth of bacterial colonies :

• can reproduce asexually very quickly

• in a closed system a build up of waste products prevents growth

• there are 4 stages in the growth curve

the stages of a bacterial growth curve :

• lag phase - bacteria are adapting to new environment, they are growing and synthesising enzymes, not at maximum reproduction rate

• log/exponential phase - bacterial reproduction at its maximum

• stationary phase - new cells and cells dying is equal

• decline/death stage - reproduction ceased and death rate increases

limiting factors which prevent exponential growth of bacteria :

• nutrients available

• oxygen levels

• temperature

• waste build up

• changes in pH

nutrients available as limiting factor of bacterial growth :

• initially plenty of food

• as numbers increase nutrients is used up

• nutrients no longer able to support further growth unless more is added

oxygen levels as a limiting factor of bacterial growth :

• as population increases demand for oxygen also increases

• therefore limited oxygen cannot support a growing culture

temperature as a limiting factor of bacterial growth :

• low temperature reduce kinetic energy and slow down reproduction

• too high temperature denature enzymes preventing the enzyme controlled reactions and killing the microroganisms s

waste build up as limiting factor of bacterial growth :

• as population increases, toxic material builds up

• this inhibits further growth and can poison the culture

changes in pH as limiting factor of bacterial growth :

• carbon dioxide produced by cells increases, causes the pH to fall

• this can denature enzymes and inhibit population growth