Cloning and Biotechnology OCR A Level, cloning and biotechnology

How does natural plant cloning occur (vegetative propagation)?

1. Bulbs

- Buds form internally which each develop into new shoots and new plants

2. Runners

- Lateral stem grows away from parent plant and roots develop

3. Rhizomes

- Specialised horizontal stem running underground, buds develop to form new vertical shoots

4. Stem tubers

- Tip of underground cell swells with stored food to form storage organ which buds to produce new shoots

define vegetative propogation

a form of asexual reproduction in which a plant grows new parts which separate from the parent - develop from meristematic regions

advantages and disadvantages of vegetative propogation

+ve:

- quicker and more certain

-identical to parent

- maintains desirable characteristics

-ve:

- overcrowding around parent plant

- little possibility of dispersal

- lack of genetic variation

Natural clones in horticulture / How to take cuttings from a plant

Splitting bulbs, removing young plants from runners, cutting up rhizomes (increase plant numbers cheaply)

Take cuttings from stem/roots/leaves

When is micropropagation used?

When desirable plant:

Does not readily produce seeds

Does not respond well to natural cloning

Is very rare

Has been selectively bred with difficulty

Is required to be 'pathogen-free'

Describe the process of micropropagation

1. Meristem tissue from shoot tips/apical buds dissected out in sterile conditions

2. Sterilise the sample using very dilute bleach (material removed from the plant is known as the explant)

3. Explant placed in sterile medium containing plant hormones to stimulate mitosis and form a callus

4. Callus divided and placed into new medium containing hormones/nutrients that stimulate growth of identical plantlets

5. Potted into compost where they grown into small plants

Arguments for micropropogation

- Allows for rapid production of large numbers of plants

- Produced disease-free plants

- Can produce viable numbers of plants after genetic modification

- Reliably increases numbers of rare/endangered species

Arguments against micropropogation

- Produces monoculture (all susceptible to same diseases or changes in growing conditions)

- Relatively expensive and requires skilled workers

- Vulnerable to infection during production process

Describe the process of artificial twinning

1. Cow with desirable trait treated with hormones to super ovulate releasing more mature ova than usual

2. Ova fertilised naturally to form a zygote and flushed out of uterus or fertilised in the lab using IVF

3. At around day 6 (cells totipotent) cells of early embryo split to produce several smaller embryos

4. Each split embryo grown in the lab for a few days then implanted into surrogate mother

5. Embryos develop into foetuses and are born naturally

Describe the process of enucleation and Somatic Cell Nuclear Transfer

1. Nucleus removed from somatic cell of adult animal

2. Nucleus removed from mature ovum of another animal

3. Nucleus from somatic cell placed into enucleated ovum - mild electric shock so it fuses and begins to divide

4. Embryo that develops transferred into uterus of 3rd animal

Arguments for and against artificial cloning

For:

- can produces clones with desirable characteristics

- increase population of endangered species

- can use to genetically engineer animals with characteristics required to make human products like cheese

Against:

- reduces variation

- can cause health problems with the clones

- shorter life expectancy

- time consuming and expensive

What is meant by biotechnology?

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

Microorganisms are ideal in biotechnology because:

There are no welfare issues

There is an enormous range of microorganisms

They can be artificially manipulated to carry out synthesis reactions

They have a short life cycle and rapid growth rate

5. Nutrient requirements are simple/cheap

Examples of indirect food production

Bread - yeast

Cheese - bacteria

Yoghurt - bacteria

Example of direct food production

Quorn - single-celled fungus grown in large fermenters

What is bioremediation?

Use of microorganisms to breakdown pollutants and contaminants in the soil or in the water

Describe use of natural organisms in bioremediation

Naturally break down organic material forming carbon dioxide and water

They can break down and neutralise many contaminants

E.g. in an oil spill nutrients can be added to water to encourage microbial growth and oil can be dispersed into smaller particles to increase SA for microbial action

Describe use of GM organisms in bioremediation

Scientists trying to develop GM bacteria which can breakdown or accumulate contaminants that they would not naturally encounter e.g. to remove mercury contamination from water

What kind of techniques are important when culturing microorganisms/ preparing nutrient medium?

Aseptic

Once agar/nutrient broth is prepared the bacteria must be added in a process called...

Inoculation

Describe the process of inoculating broth

1. Make suspension of bacteria to be grown

2. Mix known volume with sterile nutrient broth in the flask

3. Stopper with cotton wool to prevent contamination from air

4. Incubate at suitable temperature

5. Shake regularly to aerate broth providing oxygen for growing bacteria

Describe the process of inoculating agar

1. Hold wire inoculating loop in bunsen flame to sterilise it - not allowed to touch any surfaces as it cools to avoid contamination

2. Dip sterilised loop in bacterial suspension, remove lid of petri dish and make zig-zag streak across surface of agar (don't let it dig into the agar)

3. Replace lid, hold down with tape but not completely sealed so oxygen can get in preventing growth of anaerobic bacteria

Lag Phase

Bacteria adjusting to new environment, they are growing but are not yet reproducing

Log/Exponential Phase

Rate of bacterial reproduction is close to or at its theoretical maximum

Stationary Phase

Death rate is the same as reproduction rate

Death Phase

Reproduction has almost ceased, death rate is higher

Factors affecting exponential growth:

Nutrients available

Oxygen levels

Temperature

Build-up of waste

Change in pH

What are primary metabolites?

produced as part of normal growth

usually waste products

examples - proteins, enzymes, ethanol, lactate

usually collected in a continuous culture

What are secondary metabolites?

They are molecules that are not essential for growth, but they can give the microorganism an ecological advantage.

Batch culture

CLOSED

Microorganisms inoculated into fixed volume of medium

Competition for finite resources

Overall growth ceases at stationary phase - at this stage microorganisms usually carry out processes to carry out desired end product

Stopped before death phase

System cleaned and sterilised

Continuous culture

Sterile nutrient medium added continually once it reahces point of exponential growth

Useful and waste products continually removed

What are immobilised enzymes?

Enzymes that are attached to an insoluble material so they can't become mixed with the products

What are the advantages of isolated enzymes?

Less wasteful

More efficient

More specific

Maximise efficiency

Less downstream processing

What are the advantages of immobilised enzymes?

Can be reused (cheaper

Easily separated from reactants and products so reduced down-stream processing

More reliable

Greater temperature tolerance (less easily denatured by heat)

Ease of manipulation

Disadvantages of immobilised enzymes

Reduced efficiency

Higher initial cost of materials/bioreactor

More technical issues

How are enzymes immobilised?

Adsorption - enzymes bound to clay surface by hydrophobic interactions and ionic links (weak bonds so some enzymes break loose)

Covalent bonding to clay surface (can be expensive and reduce enzyme action)

Entrapment - enzymes trapped in matrix

Membrane barrier - semi permeable membrane around enzymes (limits rate of reaction)

What is a clone?

a genetically identical copy of a gene or organism

What is vegatative propagation?

Natural cloning - a form of asexual reproduction where a plant grows new parts that eventually become separated from the parent

use of meristem?

Meristem is plant tissue where growth occurs.

Meristematic cells are stem cells that divide and differentiate into any type of specialised cell.

They are totipotent

Forms of natural vegetation propagation

Parrenating bulb - leaf bases swell with stored food from photosynthesis, buds form internally and developinto new shoots next season e.g. dafodil

Parranating tuber - tip of underground stem swells from food to form tuber or storage organ and buds develop to produce new shoots e.g. potato

Runners - lateral stem grows away from parent plant and roots develop when runner touchers ground, runner withers and dies e.g. strawberry plant

Rhizome - horizontal stem running underground, buds develop and form new vertical shoots e.g. marram grass

Micropropogation (Tissue culture)

process of making large numbers of genetically identical offspring from single parent plant using tissue culture:

~ take small sample of tissue from plant you want to clone e.g. meristem tissue

~ steralise sample, material from plant = the explant

~ explant placed in sterile culture containing hormones (auxins, cytokinines) which stimulates mitosis + forms callus

~ callus divided up and placed in different culture containing nutrients = genetically identical plantlets

Advantages of micropropagation

desirable features, very quick and easy to produce large numbers, can easily reproduce rare or endangered plants, can reproduce disease-resistant strains of plants, all year round

disadvantages of micropropogation

expensive, requires skilled workers, explants and plantlets are vulnerable to infection, if infected, all clones may be lost

Natural clones in animals

2 methods used for production of high quality farm animals:

~ somatic cell nuclear transfer

~ artificial/embryo twinning

Artificial embryo twinning

separation of a developing embryo to form two independent identical clones. - but can be spilt into more separate embryos

~ female with desirable traits is treated with hormones to release many eggs

~ eggs fertilised inside female and embryos flushed out OR eggs fertilised in lab by IVF

~ embryonic cells still totipotent, so manually split to produce many smaller embryos

~ split embryos left to divide for few days

~ each embryo placed into surrogate mother

~ embryos develop into foetesus and produce many genetically identical clones

somatic cell nuclear transfer

a method of producing a clone from an adult animal by transferring the nucleus from a somatic body cell of adult animal into an egg cell

1. egg cell harvested from female and nucleus removed

(enucleation)

2. somatic cell from animal to be cloned is isolated

3. somatic cell and enucleated egg cell are joined using electrofusion

4. cell undergoes mitosis to eventually form an embryo

5. embryo then implanted into surrogate mother

6. embryo develops into fetus and clone

arguments for animal cloning

~ animals with desirable characteristics can be cloned to maximise agricultural output

~ remove less desirable charcteristics from gene pool

~ help preserve endangered species

~ provide regenerated organs - no risk of rejection

arguments against animal cloning

~ SCNT very hit-or-miss (took hundreds of attempts to clone Dolly)

~ unknown long-term side effects - high number of early deaths and genetic abnormalities

~ some can grow abnormally large - can cause breathing and circulatory problems in animals

~ cloning destroys embryos

Plant cloning method additional

Suckers- shoots that grow from sucker buds present on the shallow roots of a parent plant.

Plant cutting method

1. use a scalpel to take a cutting between 5cm and 10cm long from the parent plant.

2.remove the leaves from the lower end of your cutting so it is just one at the tip.

3.dip lower end into rooting powder which contain hormones for root formation.

4.place cutting in a pot containing a suitable growth medium.

5.provide your cutting with a warm and moist environment by either covering pot with a plastic bag or lacing it into a propogator

6. when cutting has formed roots, plant elsewhere

Animal clones can occur naturally

During sexual reproduction,once an egg has been fertilised it is possible to split during the early stages of development and develop into multiple embryos with the same genetic information.

Why are microorganisms/living organisms used fro biotechnology?

-ideal growth conditions can be created eg. ph,temp, nutrients

-short life cycle so products can be made quickly

-economical

-grown any time during the year

biotechnology

-it can use enzymes

-intracellular enzymes are contained in a cell

-isolated enzymes aren't contained within a cell

-naturally secreted enzymes are cheaper to use because it can be expensive to extract enzymes from cells

Brewing

yeast is added to a grain eg.barley and other ingredients . yeast respires anaerobically using glucose from the grain and then produce ethanol and co2 (fermentation)

baking

-yeast makes bread rise

-co2 from fermentation make sure it doesnt fall flat

cheese

The milk is pasteurised (heated to 95 *C for 20 seconds to kill off most natural bacteria) and homogen sed the tat droplets evenly distritarted through the milk)

• It is mixed with bacterial cultures and sometimes chymosin enzyme and Kept until the milk separates into solid curds and hound whey * For cottage cheese, the curds are separated from the whey, packaged, and sold.

• For most cheese, the curds are cut and cooked in the whey then stained through draining moulds or choesecloth. The whey is used for animal leads The curds are put into steel or wooden drums and may be pressed. They are left to dry, mature, and ripen before eating as the bacteria continue to act for anything from a low weeks to soverol years

yogurt

it also involves lactic acid bacteria to clot the milk and thicken it

penecillin

in times of stress, fungi from the Penicillium genus produce an antibiotic, penicillin, to stop bacteria from growing and competing for resources. Penicillin is one of the most common antibiotics used in medicine, so we produce it on a massive scale. The fungus (usually Penicillium chrysogenum) is grown under stress in industrial fermenters (see next page) and the penicillin produced is collected and processed to be used in medicine.

insulin

insulin is a hormone that's crucial for treating people with lype 1 diabetes. Insulin is made by genetically modified bacteria, which have had the gene for human insulin production inserted into their DNA (see page 220). These bacteria are grown in an industrial fermenter on a massive scale and the insulin produced is collected and purified.

bioremeditation

Bioremediation is a posh name for the process of using organisms (usually microorganisms) to remove pollutants, like oil and pesticides, from contaminated sites. Most commonly, pollutant-removing bacteria that occur naturally at a site are provided with extra nutrients and enhanced growing conditions to allow them to multiply and thrive. These bacteria break down the pollutants into less harmful products, cleaning up the area. For example, bioremediation using bacteria has been used to clean up oil spills at sea

adv of microorganisms in human food

Microorganisms reproduce fast and make protein faster than animals and plants microorganisms have e high protein content with little fat

Microorganisms can use a wide variety of waste materials including human and animal waste, reducing costs

microorganisms can be genetically modified to produce the protein required

production of microorganisms is not dependant on weather, breeding. cycles ete - it takes place constantly and can be increased or decreased to match demand

no welfare issues when growing microorganisms

Can be made to taste like anything

disadv

some microorganisms can also produce toxins if the conditions are not maintained at the optimum

the microorganisms have to be separated from the nutrient broth and processed to make the food

need sterile conditions that are carefully controlled adding to costs

often involve GM organisms and many people have concerns about eating GM food

the protein has to be purified to ensure it contains no toxin or contaminants

many people dislike the thought of eating microorganisms grown on waste

has natural flavour- needs additives

Aseptic techniques

An important part of culturing microorganisms is using aseptic techniques. These are used to prevent contamination of cultures by unwanted microorganisms, which may affect the growth of the microorganism being cultured. Contaminated cultures in laboratory experiments give imprecise results and may be hazardous to health. Contamination on an industrial scale can be very costly because entire cultures may have to be thrown away.

Below are some important aseptic techniques that you should follow when culturing microorganisms in the lab:

• Regularly disinfect work surfaces to minimise contamination.

• Work near a Bunsen flame. Hot air rises, so any microorganisms in the air should be drawn away from your culture.

• Sterilise the instrument used to transfer cultures before and after each use, e.g. sterilise a wire inoculation loop by passing it through a hot Bunsen burner flame for 5 seconds. This will kill any microorganisms on the instrument. Pre-sterilised plastic instruments should only be used once and then safely discarded.

-If you're using broth, briefly pass the neck of the broth container through a Bunsen burner flame just after it's opened and just before it's closed - this causes air to move out of the container, preventing unwanted organisms from falling in.

-Minimise the time that the agar plate is open and put the lid on as soon as possible. This reduces the chance of airborne microorganisms contaminating the culture. You could even work in an inoculation cabinet (a chamber that has a flow of sterile air inside it).

• Sterilise all glassware before and after use, e.g. in an autoclave.

• Wear a lab coat and, if needed, gloves. Tie long hair back to prevent it from falling into anything.

effect of temp on bacteria growth

1)You should be supplied with a sample of bacteria (e.g. E. coli) in broth. Using a sterile pipette, add a set volunge (e.g. 0.1 cm?) of your sample to an agar plate. Discard your pipette safely after use.

2) Spread the broth across the entire surface of the agar using a sterile plastic spreader. c Discard the spreader safely after use.

3) Put the lid on the agar plate and lightly tape it shut using two small pieces of tape.

4) Repeat steps 1-3 so that you have six plates in total.

5) Place three plates in a fridge at 4 °C and put three in an incubator at 25 °C. If you don't have access to an incubator, just leave the plates at room temperature, somewhere where the temperature is most likely to remain constant. The plates should be incubated upside down. This stops any condensation forming on the lid from dropping onto the agar.

6) Put another lidded agar plate in each of the two different temperature locations - these plates should be uncultured (i.e. you shouldn't have added any bacteria to them). These plates will act as negative controls (nothing should grow on them).

7) Leave all the plates for the same amount of time (e.g. 48 hours) then observe the results

8) If bacterial growth has occurred, you should see colonies of bacteria on the surface of the agar.

9) Count the number of colonies that have formed on each plate and record your results in a table.

10) Work out the mean number of colonies formed at each temperature.

conversion of lactose to glucose and galactose (immobilised enzyme uses)

Some people are unable to digest lactose (a sugar found in milk) because they don't produce enough (or any) of the enzyme lactase. Lactase breaks lactose down into glucose and galactose via a hydrolysis reaction. Industrially, fresh milk can now be passed over immobilised lactase to produce lactose-free milk for use in the production of lactose-free dairy products.

Production of Semi-Synthetic Pencillins

Production of Semi-Synthetic Pencillins

Pencillin is a useful antibiotic, but some bacteria have become penicillin resistant. Semi-synthetic penicillins can now be produced, which have the same antibiotic properties as natural penicillin, but are effective against penicillin-resistant organisms. Immobilised penicillin acylase enzyme is used in their production.

Conversion of Dextrins to Glucose

Conversion of Dextrins to Glucose

Glucose and glucose syrup are used in massive amounts in industry, e.g. they're used in the food industry to sweeten and thicken foods. Glucose can be derived from starchy foods, such as corn and potatoes, with the help of immobilised enzymes. Starch breaks down into dextrins (carbohydrate products), which are then broken down into glucose by the immobilised enzyme glucoamylase.

glucose to fructose

Fructose is a sugar that's much sweeter than glucose. It's used as a sweetener in food - using fructose rather than glucose means that less sugar is needed to obtain the same level of sweetness in our foods. Immobilised glucose isomerase is used to convert glucose to fructose on an industrial scale. Even though he eats your coursework and looks like a sultana, Doug is still even sweeter than fructose.

Production of Pure Samples of L-Amino Acids

Amino acids have two chemical forms (isomers) — L or D. Most amino acids utilised by the body need to be in the L form. Scientists are able to chemically synthesise amino acids, but end up with a mix of L and D forms. The enzyme aminoacylase separates them. Immobilised aminoacylase is used for the industrial production of pure samples of L-amino acids, which can be used for many purposes in the production of animal and human food, as well as in dietary supplements.