6.4 Cloning and biotechnology

what is pencillium chrysogenum?

antibiotics

what is fusarium venenatum?

single cell protein

what is lactobaciullus bulgaricus?

yogurt

what is rennin enzyme?

cheese

what is saccharomyces cerevisai?

bread and beer

what is escherichia coli?

insulin

what is biotechnology?

making use of living organisms (or parts of) in industrial processes

when was biotechnology officially named?

1919

why has there been recent advancements in biotechnology?

because of genetic technologies

what is bioremediation?

the use of either naturally occurring or deliberately introduced microorganisms to consume and break down environmental pollutants, in order to clean a polluted site

what does bioremediation require?

microbe which digest the contaminants as the conditions suit the microbe

clean soil contamination and underground water

is bioremediation carried out in-situ or ex-situ?

usually carried out in-situ but may sometimes be ex-situ depending on need

what are soya beans fermented to produce and what does it use?

soy sauce

uses yeast or Aspergillus

what are the benefits for using microorganisms in biotechnology?

avoids alternative chemical processes- which often require higher temperature and pressure

microbes are easy to culture and often digest waste

rapid reproduction increase number of useful microbes

can be run in lab worldwide

pure product which is easy to harvest

which other organisms can be used and how?

such as sheep, goats and cows which can be used to produce useful proteins

the proteins are often produced in the milk for easy harvesting

cows have also been used to synthesis human antibodies, which is isolated from their blood

what is fermentation?

anaerobic respiration by microbes

what is coagulation?

the thickening of milk due to denaturation of proteins used in yoghurt and cheese

what is rennet?

chemical containing the enzyme rennin- which causes coagulation of milk proteins (casein)

what is (kappa) casein?

it keeps casein (milk protein) soluble

what are single celled proteins?

a protein source from microbes- usually fungus

what is mycoprotein?

fungal protein

e.g. Quorn

what is yogurt?

milk that has undergone fermentation

what is yogurts raw material?

milk

what microorganisms are used to produce yogurt?

lactobacillus bulgaricus and streptococcus thermophilus

what is the process to make yogurt?

bacteria convert lactose to lactic acid which causes the milk protein to coagulate due to the acidity

the bacteria partially digest the milk making it easy to digest

what may be added when making yogurt and why?

additional microbes as probiotics to aid gastrointestinal function

what is the raw material of cheese?

milk

what microorganisms are used to produce cheese?

Lactobacillus then rennet (contains rennin)

what is the process to make cheese

pre-treated with a culture of bacteria that can produce lactic acid from lactose

once acidified the milk is mixed with rennet which coagulates the

initial coagulation from lactic acid then further coagulation of casein due to calcium ions and breakdown of kappa-casein (keeps casein soluble) by rennet. Casein precipitates of due to calcium ions

separation of curd (solid) pressed into moulds and then further treatment (may be inoculated with mould)

what is the process to make bread

raw material: flour, water, salt

microorganism: fungus, yeast- Saccharomyces cerevisiae

effect: anaerobic respiration of yeast produces carbon dioxide which makes dough rise

process required: mixing, proving (warm place for anaerobic respiration to occur) and cooking (evaporates alcohol)

what is the process to make alcohol

raw material: grapes containing fructose and glucose (wine), barley which is germinating (beer or ale)

microorganism: naturally occurring yeast (S. cerevisiae)

effect: anaerobic respiration of the sugar by the yeast to produce ethanol and carbon dioxide

what is the processes to make single cell proteins

raw material: any organic substrate e.g. waste paper or whey with sugar and nitrogen source

microorganism: Fungus: Fusarium venenatum

effect: Fungus grows on organic material, producing protein. the most common type is Quorn

what are the advantages of using microbes

protein based foods can be made many times faster than using animals or plants

can easily change rate to suit needs

very high protein content

no animal fat or chloresterol

can adjust the amino acid content (so that there are more rare amino acids)

no seasonal variation

not much land

what are the disadvantages of using microbes

some people do not want to eat food grown on waste

the proteins need isolating

high amounts of nucleic acid that needs removing

infection as also ideal conditions for pathogenic organisms

palatability- not the normal taste or texture

what is large scale lab production

it usually takes place in a specially designed fermenter

the process may be batch or continuous fermentation

growing conditions are carefully controlled to maximise yield

aseptic conditions must be maintained to prevent contamination

what variables need to be controlled for fermentation and why?

Temperature- to prevent enzymes being denatured or growth being too slow

Nutrients- carbon, nitrogen, minerals and vitamins needs for growth

Oxygen- want aerobic respiration

pH- enzyme action

Product concentration- could affect rate or reaction

Sterilisation- prevent contamination and competition

Mixing- brings substrates to organisms

how does continuous culture work?

continuous culture involves constantly topping up the materials inside the fermented whilst constantly harvesting the product

this is used for primary metabolites and gives a constant rate of production

how does batch culture work?

batch culture involves putting the microorganism under stress so it produces secondary metabolites

the process is carried out and then the fermenter is emptied to allow harvesting and restarted

what is penicillin?

modern strains of Penicillium chrysogenum have been selectively bred to be more productive

it is regarded as a secondary metabolite as it is only produced above a certain population size. this means the continuous method cannot be used

how is penicillin made into the drug?

1. the fermenter is run for 6-8 days. the culture is then filtered to remove the cells

2. the antibiotic is precipitated as crystals by the addition of potassium compounds. it is then modified

3. it is then mixed with inert substances to be administered in tablet form

what is the process to produce genetically modified insulin?

been produced by genetic engineering since 1987

human insulin gene inserted into bacterial plasmid (E.coli)

produced by continuous culture- relatively low cost and vast quantities can be produced to meet the demand

what is a culture?

a population of microbes

what is a colony?

a genetically identical group of bacteria produced from a single bacterium

what is a growth medium?

provide nutrients for microbes to grow

what is agar?

a growth medium containing C and N sources

what is an aseptic technique?

sterile technique with no contamination

what is inoculation?

the introduction of a microbe to a growth medium

what is incubation?

providing ideal conditions for growth (usually warmth)

what is an autoclave?

high temperature and high pressure environment to provide sterile equipment

what are the microbe requirements?

carbon source- yeast extract

nitrogen source- peptones from gelatine breakdown

warmth- from incubation

moisture- water in agar

no competition- from sterile technique

a growth medium will contain these things and may be in liquid broth form or jelly-like agar

what are the basic rules of aseptic technique

you must be as clean as possible (wash hands, lab coat)

work area must be sterile- disinfect, cone of sterility

equipment must be sterile (flame bottles upon opening and closing, keep lid on petri dish, flame glass/ metal equipment)

think about every action in advance

what is the 3 stage process

sterilisation- of nutrient agar and equipment

inoculation- introduction of desired microbe to the growth medium

incubation- creating conditions for microorganisms to multiply

what are the sterilisation steps?

autoclave- high temperatures and high temperatures (121^oC, 15 minutes)

used for equipment and medium

this kills all living organisms. once cooled enough the medium is poured into a sterile dish and left

what are the 3 methods of inoculation?

streaking- wire loop transfers drop onto agar

seeding- small drop of liquid culture on surface of agar or in dish before agar

spreading- glass spreader used to spread drop over agar

what are the incubation steps?

leave to settle before taping

do not seal completely- anaerobic

label with date, contents and initials

place in incubator (never above 30 degrees)

store upside down to prevent condensation drips

examine after 24-36 hours

autoclave dishes, never open

what is liquid broth?

must be aseptic

most often used to create a population before transfer to agar

serial dilutions used to allow counting of colonies

how do you measure growth rate?

serial dilutions

the number of microorganisms in a broth can be hard to count. so to investigate growth the population density must be reduced

it is normal to do this by a factor of 10.1 of broth to 9 of distilled water each time

a drop of each solution is used to inoculate agar plate. the one you can count needs multiplying back up

what is lag phase?

small population, low growth rate

adjustment phase: gene activation for metabolism, protein synthesis, cell growth

what is the log phase

this is exponential growth

enzymes, nutrients and space all present. rapid growth and reproduction. every generation doubles in size

what is the stationary phase

when nutrients are becoming limited and metabolic waste products accumulate, growth rates decline until the point that growth rate equals death rate

in this phase there is no increase in the population of live bacteria

generally in this phase bacteria produce endospores, toxins and antibiotics

what is the decline phase?

nutrients are exhausted

lethal concentration of waste products

what are primary metabolites?

collected in the log phase

maintain optimal conditions in continuous fermentation

what are secondary metabolites?

produced at end of stationary phase

closed culture

run to decline phase

what are enzymes in biotech?

both intra and extra cellular enzymes are used in biotechnology

enzymes may be isolated from natural sources or manufactured

in all cases they are reusable after the reaction

immobilisation allows the enzyme to be held outside of the reaction mixture and thus reused easily

what are the advantages of immobilisation?

easier to separate enzyme and products

increases stability and can be manipulated easily

allows continuous production/enzyme used for longer

enzyme can be recovered and reused

enzyme does not contaminate product/no purification required

what are the disadvantages of immobilisation?

immobilisation may alter shape of enzyme

may reduce catalytic ability- not freely mixing with substrate

expensive to set up, time, equipment and materials

contamination is costly

what happens in absorbsion?

where enzymes bind with immobilising support

uses a porous support

• clay, resin, glass beads

hydrophobic interactions and ionic links

enzymes can become detached (leakage) easily

active site may be distorted lowering activity

what happens in covalent bonding/cross linking?

covalent bonds link enzyme to an insoluble material

cross-linking agent used

unlikely to become detached

likely to interfere with active site

expensive to produce

what happens in entrapment?

trap enzymes in a gel bead or network of cellulose fibres

does not affect active site

reaction rates can be reduced because active site is less easily available

substrate molecules must also be able to pass through entrapment barrier

what happens in membrane separation?

enzymes are physically separated from substrate mixture by a partially permeable membrane

substrate molecules must be small enough to fit through this membrane

product molecules must also be able to pass back through membrane

active site unaffected

what are the advantages of natural cloning?

conditions that are good for the parents are good for the offspring

rapid as the population can increase rapidly

reproduction can be carried out with one parent and sexual reproduction not possible

what are the disadvantages of natural cloning?

offspring may become overcrowded

no genetic diversity (unless mutation during DNA replication)

little variation

selection not possible

whole population susceptible to changes in the environment

why can plants reproduce by cloning?

because of vegetative propagation, they have many cells that retain the ability to differentiate

what is vegetative propagation?

a method of asexual reproduction in plants where a new plant grows from a fragment of the parent plant

what are examples of vegetative propagation and how do they work?

runners- called a rhizome if underground -horizontal stem growths that can form roots at certain points

suckers- the old branch may die and the new branch replaces it

bulbs- underground system from which a group of fleshy leaves grow

corms- solid rather than fleshy like a bulb

leaves- clones grow on the leaf margins. immature plants drop off and take root

tubers- underground stem. one potato can grow into one or more plants

what is tissue culture?

growing new tissues, organs or plants from certain tissues cut from sample plants

what is micro propagation?

growing large number of plants from meristem tissue taken from a sample plant

when should micropropagation be used?

when the desirable plant doesn’t:

• produce many seeds

• doesn’t respond well to natural cloning

• rare

• GM or selectively bred

• needs to be pathogen free

what is the micropropagation process?

cells are taken from shoot (called explant)

cells are sterilised before being placed onto nutrient medium

explants placed on sterile growth medium (glucose, phosphates, amino acids, hormones)

forms a callus culture

divided to produce lots of small clumps or undifferentiated cells

transferred to a new agar medium- plantlets grow

plantlets are transferred into compost