Cloning and Biotechnology

vegetative propagation

-form of asexual reproduction

-plants produce genetically identical offshoots (clones) of themselves

-which develop into independent plants.

perennating organ

-plant structures which allow them to survive adverse conditions

-contain stored food and can remain dormant in the soil

vegetative propagation

-form of asexual reproduction

-plants produce genetically identical offshoots (clones) of themselves

-which develop into independent plants.

perennating organ

-plant structures which allow them to survive adverse conditions

-contain stored food and can remain dormant in the soil

Natural cloning examples

- Bulbs of daffodils will begin to asexually reproduce as the leaf base swells with stored food from photosynthesis

- Runners will begin to asexually reproduce as a lateral stem will begin to grow away from the parent plant and develop its own roots when it touches the ground

- Stem tubers will become swollen with stored food to form tubers (Storage organs). Buds on these tubers will grow into new shoots

Natural cloning use in horticulture

-they take cuttings from the perennating organs and split them up to grow more crops which will be cheaper

-take cuttings from many plants from their stems and grow them using rooting hormone on the base of the cutting; propagation

-more of the exact same plant; higher yield grows faster than seeds

-quality of the vegetable guaranteed

-if there is a selection pressure that occurs, due to the lack of genetic diversity all the plants will be affected

What is micropropagation?

-process of making large numbers of genetically identical offspring

-from a single parent plant using a tissue culture techniques

Why do we use micropropagation

If we need to reproduce a desirable plant but:

- it does not produce seeds

- does not respond well to natural cloning

- very rare (endangered)

- has been genetically modified or selectively bred with difficulty

- buyer requires it to be pathogen resistant

Micropropagation steps

1. Ensure surroundings are sterile as this is important so no fungi contaminate the experiment

2. Break off a small piece of the plant we are going to clone (cauliflower). Cut this into a thin transverse section - explant

3. Sterilise the explant by soaking it in ethanol or bleach for 15 minutes to ensure only cauliflower cells are present on the explant

4. Using sterile forceps , add the explant to a container of agar growth medium which will provide the nutrients for the plant to grow

5. Leave the plant to grow on a window sill for 3 weeks

Results of micropropagation

- completely grown cauliflower clone of the explant

We are able to clone plants because they are totipotent

Advantages for micropropagation

- rapid growth

- high quality

- large quantity

- helps endangered plants

- create more plants after genetic modification

Against micropropagation

- produces identical plants so more susceptible to diseases

- relatively expensive

- plantlets are vulnerable to infection

- large number of new plants are lost

Natural cloning in animals

-mammals clone when identical twins are formed

-occurs when a zygote divides as normal; two daughter cells then split to become two separate cells

-each cell grows and develops into a new individual

-Amphibians and reptiles can naturally clone without a male

-may give birth to a male showing that they are not clones of their mother

Cloning in invertebrates

-regenerate entire animals from fragments of the original if damaged, e.g. starfish

-form new identical animals as part of their normal reproductive system, e.g. flatworms and sponges

-produce small buds on the side of their body developing into identical clones, e.g. Hydra

-some insect females produce offspring without mating

2 methods in which we are able to cause cloning in vertebrates

- Artificial twinning

- Somatic cell Nuclear transfer

Artificial embryo twinning

Purposely dividing growing ball of cells. When this occurs naturally it forms twins.

- Breed a female with a good traits with a male with good traits

- The egg and the sperm from both of the animals are put into a petri dish in which they form a embryo

- The totipotent blastocytes are then manually sliced into pieces and put them into their own Petri dish

- We then put these embryos back into surrogate mothers (single pregnancies can cause less issues)

Somatic cell nuclear transfer

a cloning technique that involves substituting genetic material from an adult's cell for the nucleus of an egg

SCNT process

- Take the nuclei from a somatic cell of an desired animals (body cell from cow udder) and isolate this nuclei

- Take an egg cell from another animal and remove the nuclei from that egg . This becomes an "enucleated cell"

- Put the desired nuclei into the egg and provide a mild electric shock to promote mitosis for cell division

* sometimes we don't use an enucleated cell and instead we do electrofusion on two cells next to each other

- This cell division will form a totipotent blastocyte which can be implanted into a surrogate mother

-Genetically identical to the first animal

Arguments for animal cloning

- higher yield due to more desirable characteristics

- faster

- SCNT can be used in Pharming

- avoid mating risks

- infertile animals able to reproduce

- can help endangered species

Against animal cloning

- lack of genetic diversity

- expensive

- success rate is poor

- cloned animals have a shorter life span

- many malformations and miscarriages

UNETHICAL

Biotechnology

- The use of biological organisms and enzymes to form and synthesises things from bread and beer to insulin and antibiotics

-indirect use microorganisms to enhance food production

-direct microorganism directly prod protein you can eat

Why are microorganisms ideal to use for manufacturing INDIRECTLY

- do not take up much space

- high reproduction rate

- very short life cycle

- easier to control

- nutrients required are relatively cheap and they are low maintenance

- conditions in which they thrive are very low and affordable

Disadvantages of using microorganisms in food production INDIRECTLY

- if the conditions are not ideal, the microorganisms are unable to function and therefore do not work efficiently

- if the conditions are ideal for microorganisms to indirectly help food production , they can also be ideal for the growth of microorganisms that cause food to spoil or that cause disease meaning sterile conditions are required

- most microorganisms are GM organisms and so ethical issues on GM organisms can occur

Advantages for using microorganisms for food DIRECTLY

- reproduce fast so there is a large food supply to feed a large population

- high in protein

- can use waste materials so reduce cost

- can be easily genetically modified to produced desired qualities

- can taste like anything

- easy to grow

Disadvantages of using microorganisms to grow food DIRECTLY

- some can produce toxins if the conditions are nor optimum

- dislike idea of eating microorganisms

- people against eating GMO food

- sterile conditions required so it can be expensive

- additives needed

Why should we always take health precautions when culturing microorganisms even when they are harmless

- risk of mutations

- risk of contamination from environment

What is nutrient medium

-substance that microorganisms require in order to reproduce rapidly

-nutrients can be in broth form or agar form but when providing nutrient medium, it must be under sterile and aseptic techniques

Inoculating broth

1. Make a suspension of bacteria to be grown

2. Add a known volume of sterile nutrient medium to the suspension

3. Add a stopper on the flask to prevent contamination

4. Place in a suitable temperature and make sure to shake it to keep it well aerated for aerobic bacteria

Inoculating with agar

1. Heat inoculation loop on a bunsen burner to kill off any other bacteria that could contaminate the mixture

2. Dip the sterilised loop in the bacterial suspension and remove the lid of the Petri dish slightly

3. Make a gentle zig zag with the loop and then add a new petri dish lid ontop

4. Seal it slightly with tape to allow oxygen to enter to prevent anaerobic bacteria growth

How can populations of microorganisms be counted

- Direct counting counting all the cells you see, bot living and dead cells by taking samples

- Viable counting which involves culturing samples of microorganisms and counting only the colonies that grow

- Turbidity , using a colorimeter to measure the absorbance of both living and dead microorganisms

Turbidity

a measure of the amount of microorganisms in a broth culture depending on its cloudiness and how much light can pass through the suspension

- the more cloudy and turbid it is, the more reproduction that is occurring

Change in turbidity can be measured from initial inoculation to the end using time intervals and can then be plotted on a population growth curve

Stages of Population Growth

lag phase, exponential growth phase, stationary phase, death phase

Lag phase

population size stays on a low due to the microorganisms just adjusting to their environment and making the enzymes required for their process

Log phase

This is the exponential phase, microorganisms will exploit their resources and use them all up causing more reproduction of microorganisms and so population doubles within each division

Stationary phase

Population reaches its maximum as now there are limited resources as they have used them all up in the log phase

They now reach an equilibrium of the number of reproduction = the number of deaths

Decline phase

There is an all time low of nutrients and so death rate rises and toxic substances builds up which will cause a decline in the graph

How do microorganisms reproduce

Using binary fission

1. The circular DNA molecule will undergo DNA replication along with any other plasmids

2. the parent cell will divide into 2 identical daughter cells

3. These cells will have one copy of DNA EXACTLY THE SAME

How to calculate rate of cell division by binary fission

N=N0 X 2^n

N = the final number of bacteria

N0 = the initial number of bacteria

n = number of divisions

why are log scales used to graph bacterial growth

a tradition 1, 2, 3 scale would be unrealistic. log scales are needed to express the huge numbers of bacteria that are growing. a small macroscopic colony accounts for millions of cells and would be improbable to graph using arithimatic numbers

uneven intervals

Factors that can prevent exponential growth

- nutrients available

- low oxygen levels due to more respiration

- temperature (low temp slows down reproduction)

- build up of waste

- change in pH due to high CO2 levels

Serial dilutions for microorganisms

We create serial dilutions by taking a series of dilution from a stock solution

This allows it to be easier for us to count how many colonies are in a solution and from that we can find out how many estimated colonies are in the total solution

Bioprocess

specific process that uses complete living microorganisms or their components to obtain desired products

Primary metabolites

products of metabolism essential to survival

- ethanol in anaerobic respiration in yeast

- amino acids

- enzymes

All required for the yeasts survival

- usually formed in a period of active growth

secondary metabolites

organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism. They do not affect the organisms everyday survial

- pigments

- defence chemicals

organisms would not suffer without these for a short term

2nd metabolites are used in the process of penicillin

- tend to be formed during the stationary phase of the population graph

Types of bioprocess

Batch fermentation and continuous fermentation

Batch fermentation

- microorganisms are inoculated into a fixed volume of medium

- as growth takes place, nutrients are used up and waste is made

- this means that when the culture reaches the stationary phase , overall growth will stop and so microorganisms will go through biochemical changes to combat this lack of ideal conditions in order to form the end product

This process is stopped before the death phase and the products harvested

After this, the entire bioreacter is cleaned and sterlisied and the microorganisms are thrown away

Continuous culture

A culture system in which new medium is continuously added to replace old medium.

- waste products are continuously removed

Semi-continuous cultivation

"Cells in an actively dividing state which are maintained in culture by periodically draining off the medium and replenishing it with fresh medium"

How to control bioreactors for a maximum yield

- temperature should be controlled to prevent denaturing and to increase levels of reproduction to increase metabolite production

Sensors are in the bioreactor to detect drops in levels

- Nutrients and oxygen

- Mixing things up - bioreactor needs to constantly be mixing with paddles to ensure that everything is being equally distrubuted

-aspesis - important to prevent any contamination, bioreactor should be completely sealed

Isolated enzymes

Enzymes that aren't contained within cells

Why are isolated enzymes better than microorganisms

- less wasteful

- even lower maintenance as they do not need nutrients

- more efficient as they can work at much higher concentrations

- no wasteful by products are made as there are no unwanted enzymes

- higher yield

What can be an isolated enzyme

intacellular and extracelluar enxyme sboth made from a microorganism

Advantaged of extracellular enzymes

- secreted outside the microorganisms so easier to extract

- microorganisms produce only a few extracellular enzymes making it easier to separate and identify uses for each

-extracellular enzymes can survive better in a variety of different conditions that intracellular due to intracellular enzyme conditions being very constant

disadvantage of extracellular

- as more intracellular enzymes are made, they are more likely going to be more ideal for the process needed

Uses of intracellular enzymes as isolated enzymes

- used for food preservation with glucose oxidase

- asparaginase for cancer treatment

- penicillin acylase for conversion of natural penicillin to semi synthetic drugs(more effective)

Immobilised enzymes

Enzymes that are attached, or fixed, to each other, or to an inert material

mimicks how an enzyme is in a cell as its membrane bound

This allows more and more catalytic processes to happen without it being replaced unlike free enzymes which constantly get lost and must be replaced

More economical

Advantages of immobilised enzymes

- reusable

- less downstream process so cost friendly

- greater temperature tolerance

- more reliable due to the amount of control we have over them

- able to be easily manipulated which can keep bioreactors continuosly going and therefore costs are low

Disadvantages of Immobilised enzymes

- high inital cost

- reduced efficiency as imobblising enzymes may reduce its activity rate

- more technical issues as reactors that use immobilised enzymes are more complex and expensive so more things can go wrong

How can enzymes be immobilised

- Adsorption to an inorganic carrier

- Entrapment in a matrix

- covalent or ionic bonds to inorganic carrier (stronger than adsorption)

- encapsulation

Pro and cons Adsorption

PRO:

- Simple and cheap

- enzymes are very accessible to substrate

- universal

CON:

- due to weak bonding, enzymes can be lost very easily

Pros and cons adsorption with covalent or ionic bonding

PRO:

- Less likely to be lost

- pH and sub conc have little effect on enzyme

- accessible

CONS:

- expensive

- due to the strong ionic bonds, active site may change being no longer complementary

Pros and cons of entrapment

-PROS

- universal to many different processes

CONS:

- expensive

- difficult to entrap

- diffusion of substrate to get inside the capsule and out will take time

Encapsulation pros and cons

PROS:

- simple

- small effect on enzyme activity

- universal

CONS:

- diffusion takes time

- expensive