cloning and biotechnology

what is an immobilised enzyme

an enzyme which is attached to an unreactive, insoluble material

what is an extracellular enzyme

an enzyme which is secreted by a cell and functions outside of the cell e.g. digestive enzymes

what is an intracellular enzyme

an enzyme which functions within the cell in which it was produced e.g. catalase

what are the four methods of immobilisation

• adsorption - enzymes may be adsorbed onto the surface of insoluble support materials e.g. collagen or resin

• covalent association - enzymes may be bound to insoluble support materials e.g. cellulose, collagen fibres by covalent or ionic bonds

• entrapment - enzymes may be trapped in a matrix e.g. silica gel

• encapsulation - enzymes may be isolated by a partially permeable membrane

advantages of using immobilised enzymes

• they are easily separated from the product, so are recoverable and recycled as opposed to free enzymes which mix into solutions and are often wasted

• they do not contaminate the product so there is less ‘downstream processing’ therefore they are cheaper to use and more time efficient

• improved stability from the substance they are immobilised by so they are more tolerant of temperature and PH changes than free enzymes

disadvantages of using immobilised enzymes

• they can reduce activity rate as they are not free to move, so there are fewer successful collisions

• high set up costs for materials and a bioreactor than there is for free enzymes, so are not always cost-effective for small scale productions

give examples of the use of immobilised enzymes and it what industry they are used in

• lactase - for the hydrolysis of lactose to glucose and galactose (dairy industry)

• glucose isomerase - for the conversion of glucose to fructose (food and beverage)

• penicillin acyclase - forms semi-synthetic penicillin’s (pharmaceutical - trying to avoid antibiotic resistance)

• aminoacyclase - produces pure samples of L-amino acids (pharmaceutical)

• glucoamylase - catalyses the conversion of dextrins to glucose (starch processing industry)

• nitrilase - catalyses the conversion of acrylonitrile to acrylamide (plastics industry)

explain the differences between free/soluble and immobilised enzymes in reference to optimum temperature

higher optimum temperature for the immobilised enzymes as they are more tolerant to higher temperatures and so are less easily denatured. this is due to the element of protection from the material they are bound to.

explain the differences between free/soluble and immobilised enzymes in reference to maximum activity rate

the max activity rate is higher for the free/soluble enzymes as they are free to move, so there are more successful collisions between the enzymes and the substrate (so more enzyme-substrate complexes are formed). immobilised enzymes are attached to a material, so not free to move, and so less successful collisions occur.

what are biosensors

• they detect the presence of/concentration of specific biological molecules

• they convert a biochemical reaction into an electrical signal (a transducer) that can be further amplified, processed and recorded

• e.g. pin prick tests for diabetes (glucose)

what is asexual reproduction

the production of offspring that are genetically identical to the parent, the offspring are referred to as ‘clones’

identify some methods of asexual reproduction in plants

• rhizomes - specialised, underground stems, they store food and can produce new vertical shoots and roots from buds on nodes along the rhizome e.g. marram grass

• stolons - specialised stems that grow along the soil surface away from the parent plant, with nodes or stem tips that can root to form a new plant upon contact with the ground e.g. strawberries

• suckers - shoots that emerge from the shallow root buds of the parent plant e.g. elm trees

• tubers - form when the tip of a stem becomes swollen with food, with buds on the tuber surface that can develop into new shoots e.g. potatoes

• bulbs - form when a leaf base becomes swollen with stored food, and the bud inside the bulb can form new shoots e.g. daffodil

how to take a cutting (artificial cloning in plants)

• choose a healthy stem, remove it from the parent plant by cutting between nodes at a slanted angle (gives a larger surface area)

• remove the lower leaves so there is a bare stem (they would use energy)

• dip into a rooting powder - a mixture of auxins and fungicides

• place the cutting into a moist soil and cover with a plastic bag to reduce water loss (transpiration)

• once rooted, transplant the new clone
  

what is an explant

a small sample of meristem cut from a parent plant

what is a plant calluses

a growing mass of unorganised and undifferentiated plant parenchyma cells, they cover a plant wound

what are plantlets

young plants which can be removed and grown separately

describe the process of micropropagation

• small amounts of tissues or cells are taken from a plant (explant which contains totipotent cells)

• the cells are then transferred to plates that are filled with sterile nutrient agar jelly

• auxins are then added to prepare all the cells for mitosis

• small masses of tissue grow at a fast rate (callus)

• growth hormones are then added to prepare the long process of root and stem differentiation and growth

• the tiny plantlets are then put into potting trays where they develop into an adult plant
  

advantages of asexual reproduction

• it is fast

• it ensures a high yield

• it is cost effective

• it maintains the quality of the crop as the new plants have the same genetic traits as their parents

disadvantages of asexual reproduction

• it results in a lack of genetic variation in offspring

• the plants are more susceptible to disease, pests and climate change

what are some advantages of micropropagation

• produces plants which are genetically identical so there is a reliable inheritance of traits

• it can be carried out at all times of year

• it rapidly produces a large number of mature plants

what are some disadvantages of micropropagation

• crops are more vulnerable to diseases and environmental changes

• it may unintentionally propagate undesirable traits

• it is expensive and requires skilled technicians, making it less feasible on a small scale

describe sexual reproduction

the offspring are produced by the fusion of the male and female gametes

advantages of sexual reproduction

• high genetic variability

• ‘speeds up evolution’

disadvantages of sexual reproduction

• energy costly

• courtship is time/resource consuming

describe the process of artificial embryo twinning

• a female organism is treated with hormones to produce multiple ova (egg cells)

• the ova are extracted and fertilised in a Petri dish to produce an embryo

• the embryo divides into several cells and, while the cells are still totipotent, the embryo is split

• each cell is placed into its own Petri dish to develop into individual embryos

• the embryos are implanted into the uteruses of surrogate mothers for development

what is the result of artificial embryo twinning

genetically identical animal clones

describe the process of somatic cell nuclear transfer

• a somatic cell (donor cell) nucleus is removed from an adult animal

• an ovum (egg cell) of a different female animal of the same species is enucleated (the nucleus is removed)

• the nucleus from the somatic cell is transferred into the enucleated ovum

• the somatic nucleus is fused with the enucleated ovum, often stimulated by an electric shock through electrofusion

• the fused cell begins dividing, forming an embryo

• this embryo is implanted into the uterus of a surrogate mother

• the surrogate eventually gives birth to a clone of the somatic cell donor

what are the two methods of artificial cloning in animals

• artificial embryo twinning

• somatic cell nuclear transfer

applications of animal cloning

• medical research - cloning produces genetically identical animals for drug testing and disease modelling

• conservation - can increase the number of endangered species from a limited gene pool

• agriculture - it can replicate animals with desirable characteristics for selective breeding to improve product quality

• stem cells - cloned embryos provide a source of immunocompatible stem cells for tissue repair

advantages of animal cloning

• preservation of endangered species

• reproduction of desirable traits

• revival of deceased pets or valuable animals

disadvantages of animal cloning

• low success rate

• health problems - cloned animals can suffer from abnormalities, premature growth, weakened immune system etc

• high cost

what is biotechnology

technology which exploits living organisms or enzymes to improve food science and medical science

what are primary metabolites

substances that are produced in processes that are essential for normal microbial functioning e.g. ethanol from aerobic respiration in yeast

what are secondary metabolites

substances that are produced in non-essential processes e.g. antibiotics or plant defence chemicals

what are some of the typical components of a bioreactor

• a stainless steal vat; prevents microbes from becoming trapped and corrosion of surfaces

• water jacket for circulating cooling water to remove the heat generated by metabolic activity of the micro-organisms

• sterilised nutrient solution inoculated with a pure culture of the micro-organism

• impellers (stirrers) for mixing the micro-organisms and nutrients and distributing the heat

• sparger for forcing air into the medium

• probes to monitor PH, temperature and dissolved oxygen

• ports for adding ingredients and removing products

• a sterilisation system, e.g. a steam injection

• exhaust (removes carbon dioxide produced in respiration)

what is aseptic technique

a procedure used by staff in biotechnology, used to prevent the spread of infection or introduction of unwanted organisms into specimens or the lab.

what is a culture

a growth of microorganisms, can be a single species (pure culture) or a mixture of species (mixed culture)

what factors may affect yield of microorganisms within a fermenter? and why would they have an effect

• nutrient availability - nutrient demand may exceed nutrient supply which could mean a smaller yield of micro-organisms grow

• PH - a build up of carbon dioxide may reduce PH, which can inhibit enzyme activity, so maintaining a constant PH allows microbial enzymes to function efficiently

• temperature - if it is too low, bacterial enzymes wont work so yield will be low, however if too high then bacterial enzymes will denature

• contamination - if microorganisms grow which we don’t want, there will be competition for resources against the micro-organisms that you do want

what are the two main methods for operating fermenters

• batch fermentation

• continuous fermentation

describe batch fermentation

• microbes are grown in a fixed volume in individual batches until nutrients deplete and waste accumulates

• each batch is followed by emptying and cleaning of the vessel before starting the next batch

describe continuous fermentation

• involves continuously supplying fresh nutrients at a fixed rate and removing the culture broth continuously

• this maintains the growth of the culture indefinitely

describe the four key stages in a standard growth curve

• lag phase - birth rate = death rate, cells have slow initial growth as they adapt to their environment and produce essential enzymes

• log/exponential phase - birth rate is greater than death rate, rapid doubling of cell numbers occurs under ideal conditions, and growth rate is at its maximum

• stationary phase - birth rate = death rate, growth rate plateaus as nutrients diminish and waste accumulates (so there is competition for nutrients)

• death/decline phase - birth rate is less than death rate, due to resource limitation and build up of toxins

describe the process of culturing microorganisms in a lab

• sterilise all equipment before use e.g. hold wire inoculating loop in a Bunsen flame

• dip the sterilised wire inoculating loop into a broth that contains a bacterial suspension

• transfer the microbes into a petri dish containing a sterile nutrient medium by lightly zig-zagging the loop across the agar

• close the plates and lightly tape them so they are not completely sealed

• label the plates with relevant information

• incubate the plates upside down under the required conditions

• repeat steps 1-6 with a control agar dish with no bacteria

• asses microbial growth by observing colony formation on the agar

what are some factors which may affect microbial growth and how can they be investigated

• temperature - incubate duplicate plates at different temperatures

• PH - add buffer solutions to the agar to maintain different PH levels

• nutrient availability - prepare agar with varying nutrient conditions

• antimicrobial substances - add different microbial compounds to the agar plates