Cloning and biotechnology

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what is recombinant DNA

DNA that is formed by combining genetic material from two or more different sources

usually involves inserting a gene from one organism of DNA to another

eg:human insulin gene being inserted into bacterial plasmid

applications of recombinant DNA

vaccines, genetcially1 modified crops, gene therapy, insulin production

what is genetic engineering

deliberate modification of an organisms genetic material using biotechnology

involves adding or removing genes

genetic engineering process

isolation of DNA - dna containing the desired gene is extracted

cutting DNA with restriction enzyme - dna is cut at specific sequences

cutting the vector - vector is usually a plasmid is cut with restriction enzyme (vector is a dna molecule used to carry foreign dna into a host cell)

dna ligase joins gene and plasmid to form recombinant plasmid

recombinant plasmid is inserted into bacteria bacteria

bacteria containing recombinant plasmid are selected and bacteria multiply and express inserted gene

what is pcr useed for

Produce large quantities of specific fragments of DNA

3 steps of pcr

Denaturation - double stranded DNA is heated to 95° which breaks hydrogen bonds that bond the two DNA strands together

Annealing - the temperature is decreased to between 50-60° so that primers can bind to the ends of single strands of DNA.

Elongation / Extension - the temperature is increased to 72° for at least a minute as this is the optimum temperature for Taq polymerase to build complementary strands of DNA to produce new identical double stranded DNA molecules

what is molecular cloning

process of making multiple identical copies of DNA

example of molecular cloning

cloning the human insulin gene

The insulin gene is isolated from human DNA.

Both gene and plasmid are cut using the same restriction enzyme.

DNA ligase joins them.

recombinant plasmid enters bacterial cells

bacteria with recombinant plasmids are identified using antibiotic resistance markers

bacteria reproduce, copying insulin

sanger sequence

DNA template prepared

primer added

DNA synthesis occurs

random incorporation of ddNTPs stops synthesis

Fragments separated by electrophoresis

sequence determined

what is gene therapy

treatment of disease by introducing, removing, or altering genetic material in a patients cells.

how does gene therapy work

identify defective gene

normal copy of gene is inserted into cells

cells produce correct protein

what are the delivery methods

viral vectors - (retroviruses) - carry genes into cells

They produce genetically identical copies of themselves through asexual reproduction (mitosis)

How do plants eg: strawberries

Low genetic biodiversity meaning they can get disease easier

Problems with uses clones

Use a healthy shoot/ cut shoot from healthy plant Cut stem at slant between nodes Dip end of the stem in rooting powder/auxin Place in soil and add water To reduce transpiration cover with a plastic bag/ remove some leaves

How can we produce clones from cuttings

joining the shoot of one plant to the growing stem (cut at an angle to increase surface area of adhesion) and root of another plant

What is grafting

Cheap Quick compared to seeds and can have large yield Can clone seedless fruits Clones can have desired features

Advantages of cloning

Low genetic biodiversity meaning they can get disease easier Can be a labour intensive expensive process

Disadvantages of cloning

how do we do micropropagation through tissue culture

Start with carrot or cauliflower for example

Aseptic conditions (sterilise area where working and equipment)

Cut plant material into explants eg: leaf or root or bud

Sterilise explant with bleach or alcohol

•Place explant on agar containing glucose and amino acids

•A callus will form (from mitosis)

•The callus subdivides and treat to induce roots by using Auxin and cytokines or change plant to hormone ratio

•You put it in soil in water then into a greenhouse

•Desirable genetic characteristics •Tissue culture means plants can be reproduced any season because environment is controlled •Produces plants quickly and cheaply •Doesn't require a lot of space •Used to reproduce seedless plants

Advantages of artificial plant cloning (micropropagation of tissue culture)

Undesirable characteristics can be passed on Low generic diversity means they can be easily wiped out

Disadvantages of artificial plant cloning (micropropagation of tissue culture)

•Starfish regenerating limbs •Asexual reproduction of flatworms to make identical copy of themselves •Hydra which can produce clones on side of body through asexual reproduction

Examples of natural clones

Genetically identical twins Depending on what day the fertilised egg splits depends on if the twins will share placenta or a sac

What is natural cloning in humans

How do animals produce identical twins

An egg is fertilised by sperm

This forms a zygote

Zygote undergoes a few cell divisions to become an embryo

Embryo splits into two

Two embryos that form are identical resulting in identical offspring

An organism that has a desirable trait eg: cows may have more meat or more milk yield

•That organism would be given hormone treatment to release more eggs

•The eggs are fertilised •smaller zygotes form

•it's then implanted into surrogate

Mother gives birth yo identical twins

What is artificial twinning and how does it work

Nucleus is taken from a diploid somatic cell (adult cell) (sheep 1)

•Take an egg cell from another organism (sheep 2) from same species but remove nuclei so empty ovum

•Put nuclei in enucleated egg and provide a mild electric shock to promote mitosis

•A pluripotent blastocyst is made and which is implanted in surrogate mother (sheep 3)

The offspring will be a clone of sheep 1 but it will contain genetic info from sheep 2

What is somatic cell nuclear transfer (dolly the sheep)

Desirable characteristics Faster Avoid mating risks

Advantages of animal cloning

Expensive No genetic variability so are affected easily by environmental changes Shorter lifespan Success rates are poor Ethical issues

Disadvantages of animal cloning

What is therapeutic cloning

Technique used to clone cells to replace dead or damaged cells eg: replacing brain tissue in Parkinson’s

Uses of biotechnology

Why are microorganisms used in large scale industrial processes

Rapid life cycles

Can be genetically engineered

Can use low temperature

Separate products easily

Advantages of using microorganisms for human consumption

Quick and easy to grow large numbers

Grown anywhere at anytime and can be grown on waste products

Low fat high protein products

Inputs are cheap eg: glucose oxygen ammonia etc

More efficient than animals at converting energy into biomass

Can be genetically modified

Disadvantages using microorganisms for human consumption

People may not like the fact food is produced by microorganisms

A fermented contaminated by other bacteria could lead to a ruined product

Expensive

As microorganisms reproduce quickly they might mutate

Fermented products are high in Nucleic acids which may cause harm to humans

Bacteria may be infected by viruses which ruins the product

When growing microorganism in the industry what do you need

Fermenter or bioreactor (sterile and aseptic so no contamination)

What factors have to be monitored in the growth of bacterial colonies

Temperature

Nutrients available

oxygen levels

Change in pH

Build out of waste

Diagram of fermenter

Why is asepsis important

To avoid unwanted microbes

Decrease yield of product

Contamination of product

Change conditions in fermenter

Watch is batch and continuous fermentation

Batch = microorganisms are grown in batches in fermentation vessel, once culture cycle is finished product is removed and fermented is cleaned and new batch is grown (closed culture)

Continuous fermentation = Microorganisms are continually grown and products are harvested

Nutrients are added and waste is removed throughout the culturing process

Compare batch and continuous culture

Batch = growth rate is slower, easy to set up and maintain, if contamination occurs only one batch is lost, less efficient, useful in production of secondary metabolites.

Continuous = growth is higher/quicker, set up and maintaining can be difficult, if contamination occurs huge batches are lost, more efficient, useful for production of primary metabolites

How to culture microorganisms in lab

Pour plate - where bacteria is mixed with warm agar, placed on sterile plate, mixed, and plate is incubated so bacterial colonies grow (Used to identify number of colony forming units)

lawn plate - suspension of bacterial cells is placed on plate and is spread by spreader, allows to identify colonies genetically engineered

Streak plate - to separate colonies of bacteria, dip rod into sample and scrape across a side of plate and sterilise with flame and keep repeating 4 times on other sides. After incubation you get single colonies formed.

What happens if plates are not incubated at 35°

Could lead to growth of human pathogens

Why do you flame the neck of tube

Kills bacteria at neck of tube

Why is lid held above dish when adding solution

Avoid contamination from air

When incubating why is plate kept upside down

Prevents agar from drying out

What is an immobilised enzyme

An enzyme attaches to an insoluble material to prevent it mixing with the product

Ways an enzyme can be immobilised

Encapsulation - enzyme trapped in semi- permeable membrane

Entrapment - enzyme trapped in silica gel matrix

Cross linked - covalent and ionic bonds between amino acids of enzyme

Carrier bound - enzyme is attached to a carrier eg: carbon

Advantages of immobilised enzymes

Can be reused multiple times cost effective

Greater tolerance to pH and temperature changes so more stable

No enzyme in product so no need to filter end product

Disadvantages of immobilised enzymes

Specialist expensive equipment is required

More costly to buy immobilised enzymes

Rate of reaction is sometimes lower

More complex procedure

Populations of microorganisms can be measured in 3 ways what are they

Direct counting - includes all cells both living and dead and involves taking samples to count microorganisms

Viable counting - involves culturing samples of microorganisms and counting colonies

Turbidity - measure of living dead microorganisms in solution using a colorimeter for absorbance

4 phases of standard growth curve and what happens in them

Lag phase - reproduction rate of bacteria is higher than death rate adjusts to new environment

log/exponential phase - reproduction rate is much higher because of nutrients and plenty of space than death rate

Stationary phase - occurs when total growth is 0k population is at maximum. reproduction rate and death rate are similar

Death phase - death rate is higher than reproduction rate due to lack of nutrients and toxic build up

How do you calculate number of individual organisms

N= N0 x 2^n

N0 = initial number

n = number of divisions/generations

Why is the log scale a good scale to use

Wide range of numbers fit on to the same scale