A LEVEL: Biology (F215), Biotechnology

Biology F215

Biotechnology and Gene Technology

CLONES IN NATURE

Clones: organisms with the same genetic material because they are from the same original DNA. 

mitosis: asexual reproduction in eukaryotes. Binary fission occurs in prokaryotes When there are genetic differences it is a result of mutations.

ADVANTAGES (of asexual reproduction)	DISADVANTAGES (of asexual reproduction)
quick, organisms reproduce rapidly and are able to take advantage of resources in the environment	no genetic variety, parental weaknesses will be present in all the offspring.
can be an alternative to sexual reproduction
offspring have the same genetic information to survive in their environment

Natural Vegetative propagation in plants:

production of structures in an organism that can grow into new individual organisms - genetic clones

English Elms:

When the main trunk is destroyed around two months after this, root suckers or basal sprouts appear. 

Grown from the meristem tissue in the trunk close to the ground, where there is most likely the least damage. 

suckers grown in a clonal patch in order to expand to increase take in of resources.  

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ARTIFICIAL CLONES AND AGRICULTURE

We can artificially propagate plants:

1. grafting

short section of a woody plant is joined to an already growing root (rootstock), the graft grows and is genetically identical to the parent plant but the rootstock is genetically different.

1. tissue culture 

• separation of cells of any tissue type and their grown in or on a nutrient medium, in plants the undifferentiated callus tissue is grown in nutrient medium containing plant hormones that stimulate the development of the complete plant. 

Micropropagation by callus tissue culture

1. tissue from desired plant is taken away to be cloned, this is called micropropagation, this is called an explant
2. placed into a nutrient growth medium: auxin/cyokinins/giberrellins 
3. cells divide but do not differentiate they form a mass of undifferentiated cells called a callus
4. single callus cells are removed and placed into a new growth medium which promotes shoot growth 
5. shoots are then transferred to a different growth medium promoting root growth
6. growing plants are then transferred to a greenhouse to be acclimatised, grow further then planted outside. 

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ANIMAL CLONING

Only embryonic cells are able to go through stages of division to create a new individual - totipotent close (capable of differentiation)

Two Ways:

1. Splitting Embryos

As the embryo divides, individual cells can be extracted, these cells are then able to differentiate separately to create genetically identical individuals

1. Nuclear Transfer

A differentiated cell is taken from an adult (eg.skin cell), the nucleus is removed and it is placed in an empty ovum, (enucleate ovum)

The egg goes through the normal stages embryonic development: - splitting and each new cell has the same genetic material as the one where the nucleus was extracted. 

What is non-reproductive cloning?

using cloned cells to generate new cells, tissues and organs. 

• less risks of rejection
• cloning and cell culture techniques mean that waiting for donors and organ transplants could end
• they could be the cure to diseases and accidents
• totipotent and can be used to generate any cell type
• less dangerous than major operations

ALSO:

• regeneration of heart muscle cells after a heart attack
• repair of nervous tissue for diseases like MS
• repair of the spinal cord,

also known as therapeutic cloning - many ethical problems

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BIOTECHNOLOGY

Technology based on biology and the exploitation of living organisms or biological processes to improve agriculture, animal husbandry, food science, medicine and industry. 

Using microorganisms in Industry

• grow quick at optimum conditions, 
• produce proteins/chemicals outside into the surrounding medium, which can be harvested.
• genetically engineered to specific products
• good at low temperatures = lower economic costs
• grown anywhere, unaffected by climate
• products are pure and no reactive
• grown using waste products

The Growth Curve 

< — Graph shows the population curve in a closed environment -

this means that all the conditions are fixed and contained. 

Fermentation and Fermenters

• culturing of microorganisms both aerobically and anaerobically in fermentation tanks.

Metabolism is a process, Metabolites are the products

Primary metabolites

Substances produced by organisms during their natural growth; amino acids, proteins, enzymes, nucleic acids, ethanol. 

production of primary metabolites matches the population growth.

Secondary metabolites

Not part of its normal growth. The secondary metabolites produced are antibiotic molecules The production of secondary metabolites begins after the main growth period of the orgaism. It does not match the growth in population of the organism.   

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Commercial applications of biotechnology. 

Industrial scale fermenters is essentially a huge tank which may contain loads of litres.

The growing conditions in it can be manipulated and controlled - for best yield 

Growing conditions:

• temperature 
• type and time of addition of product (carbon, nitrogen & any other nutrients)
• oxygen concentration
• pH level 

Batch Culture 

• microorganism starter population is mixed with a fixed quantity of nutrient solution
• it grows over a certain period of time, 
• no further addition of nutrients
• products are then removed and fermentation tank is emptied

(penicillin is harvested using batch culture)

Continuous Culture

• nutrients are added to the culture at regular intervals
• products are also removed at regular intervals, or continuously
• eg. insulin

Asepsis: absence of unwanted microorganisms

nutrient mediums in the fermentation tanks can also lead to supporting unwanted microorganisms and contamination.

unwanted microorganisms:

• compete
• reduce yield of useful products
• spoilage of the products
• toxic chemicals
• destroy the culture microorganism and their products

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INDUSTRIAL ENZYMES

Why enzymes are useful:

• specificity: enzymes can only catalyse with specific chemicals - leads to less by products
• temperature: enzymes are able to work at lower temperatures - cheaper economic costs
• extraction of enzymes is known as downstream processing

Immobilising Enzymes

Whilst the enzymes are able to catalyse the enzyme controlled reaction but do not mix with substrates freely. 

ADVANTAGES	DISADVANTAGES
Enzymes are not present with products so down streaming processing is cheaper	immobilisation takes additional time, equipment and materials and is more expensive
immediately available for reuse, better for continuous processes	immobilised enzymes are less active, do not mix freely with substrate
immobilised enzymes are more stable, immobilising matrix protects the enzyme molecules	any contamination is costly to deal with because the whole system needs to be stopped.

Methods for immobilising enzymes:

Adsorption

molecules are mixed with immobilising support and bind to it - combo of hydroponic interactions and ionic links. 

good: if held so that active site is exposed, adsorption has high reaction rates

bad: forces are not strong, so enzyme can detach

Covalent Bonding

covalently link (gluteraldehyde) an enzyme and an insoluble support. 

good: strong binding, little enzyme leakage

bad: can not immobilise a large quantity of enzymes

Entrapment

enzymes can be trapped - cellulose fibres - 

good: enzymes are trapped in their natural state

bad: reaction rate decrease, as substrate molecules cannot get through the trapping barrier - active site is less likely to  be available

Membrane Separation

physically separated with a from substrate mixture by a partially permeable membrane

good: substrate molecules are small enough to pass through membrane, so reaction can occur. product molecules are also small enough to pass back through the membrane. 

STUDYING WHOLE GENOME

Genomics: Study of the whole set of genetic information in the form of the DNA base sequences that occur in the cells of organisms of a particular species. The sequenced genomes are placed on public access databases. 

Advances in science that used our understanding of DNA structure to be able to:

• be used in DNA profiling
• genomic sequencing and comparative genome mapping
• genetic engineering
• gene therapy 

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The Genomic Age

Organisms all have genes: code for polypeptides & amino acids.

coding DNA only makes up for 1.5% of the coding polypeptides. 

the non-coding DNA is not junk, it may regulate different functions which we do not know about. 

Sequencing the genome 

Sequencing reactions: only works on 750 base pairs. 

Genome must be broken up into fragments. 

Genomes are first mapped to find out where the chromosome comes from. We can use micro satellites.

SHOTGUN APPROACH: SHEARING OF GENOME. 

sections are broken into BACs (bacterial artificial chromosomes) and transferred into e.coli bacteria and the cells grow and reproduce and are called clone libraries. 

Sequencing BAC section:

1. cells with specific BAC section are taken and controlled. DNA extracted from the cells and restriction enzymes are used to cut it into smaller fragments. Different restriction enzymes = different fragment types.
2. fragments are separated in electrophoresis
3. fragments are sequenced
4. computer programmes map the overlapping regions from the cuts, and reassemble the whole BAC segment sequence. 

Comparing Genomes - comparative gene mapping

we know the sequence of bases in a gene and compare genes for the same proteins across a variety of organisms.

Useful for:

• find out which proteins are of relative importance 
• evolutionary relationships
• the effects of changes to DNA/Genes
• comparing genomes from pathogenic and similar but nonpathogenic organisms to identify similar base pair sequences. 
• DNA from individuals can be analysed. 

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DNA MANIPULATION - SEPARATING AND PROBING

Electrophoresis: separates DNA into fragments depending on their size. 

• uses a gel ‘slab’ containing agarose, (covered in buffer solution)
• electrodes are attached to each end of the gel
• current passes through it

Separation of different strands is possible because:

• longer strands of DNA are caught up in the agarose gel and slowed
• shorter strands move more quickly in the gel. 
• DNA has phosphate backbone (PO4- Charge - moves towards the cathode)

Southern Blotting

• nylon/nitrocellulose sheet is placed over the gel, covered in paper towels, pressed and left overnight
• DNA fragments are transferred to the sheet and can now be analysed

DNA fragments can not been seen on the sheets. 

• label it with a radioactive marker
• place photogenic film over the nitrocellulose sheet 
• you will now see the position of the DNA samples

DNA probes

short single stranded DNA (50-80 Nucleotides)

complementary to the strand which is being investigated 

• use a radioactive marker (32-phosphate) - phosphoryl group so the location is revealed to photographic film
• fluorescent markers which show a colour when exposed to UV light. 

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SEQUENCING AND COPYING DNA

Polymerase Chain Reaction: Artificial DNA replication

useful because samples of DNA can be amplified. ——> eg. multiplied 

It works because of:

• it has an antiparallel backbone
• made of strands with a 5’ (prime) and a 3’ (prime) end
• grows only from the 3’ end
• bases pair up with complimentary base pairing rules

different to normal DNA division

• replicates are short strands/sequences of DNA
• addition of primer molecules are required to start the process
• cycle of heating and cooling

PCR IS A CYCLIC REACTION

• DNA sample mixed in a solution of free DNA nucleotides & DNA polymerase
• Heated up to 95 degrees —> breaks H-bonds —> become single strands 
• Short lengths of single stranded DNA are added, called primers 
• Cooled to 55 degrees, primers bind - forming double stranded DNA at either end
• DNA polymerase binds together the short strands
• Heated up to 72 degrees for DNA polymerase —> free nucleotides bind to the end
• DNA polymerase reaches the other end and a new double stranded DNA is generated 
• process can be done multiple times. 

 AUTOMATED DNA SEQUENCING - BASED ON ELECTROPHORESIS AND PCR

steps:

• Similar to PCR, the reaction mixture contains enzyme DNA polymerase, lots of the DNA fragments, free nucleotides and primers
• primer anneals to the 3’ prime, so DNA polymerase is able to attach
• DNA polymerase adds free nucleotides - base pairing rules
• modified nucleotide added - DNA polymerase binding site is changed, it is thrown off 
• reaction stops at that template strand 
• this goes on for a while, but the final added nucleotide is tagged with a specific colour
• it is run through a machine and the sequence of colours/bases can be read.

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INTRO TO GENETIC ENGINEERING 

The processes in genetic engineering are often called recombinant DNA processes. 

The necessary steps for genetic engineering:

• required gene obtained
• copy of gene in a vector
• vector carries the gene to the recipient cell
• recipient expresses the gene through protein synthesis

Stage in Engineering Process	Methods Possible
Obtaining the gene to be expressed	mRNA which transcribed the gene can be found in cells where that gene has been expressed. eg. insulin gene can be found in the beta cells in the islets of langerhans gene can be synthesised using an automated polynucleotide sequencer  DNA probe can locate the gene on DNA fragments, the gene will then be cute using restriction enzymes
Placing gene into a vector	sealed into a bacterial plasmid - using enzyme ligase  genes can be sealed into virus genomes the vectors may have to contain regulatory sequences of DNA - this means the transcribed gene is able to fit into the host cell
Getting the gene into the recipient cell	it depends on the cell but: electroporation -> high voltage pulse to disrupt the membrane microinjection -> DNA is injected with a micropipette into the cell viral transfer -> vector is a virus, uses virus’ mechanism for infecting cells Ti plasmids -> inserted into agrobacterium tumefaciens, plants are infected with the bacteria which inserts the DNA into the plant’s genome Liposomes -> wrapped in lipid molecules - fat soluble and are able to cross lipid membranes by diffusion

restriction enzymes cute DNA backbones, ligase seals them 

restriction enzymes: cut at specific points (50 commonly used restriction enzymes)

• will cut only where specific base sequence occurs - restriction site 
• it is less than 10 bases long
• they catalyse a hydrolysis reaction - breaks the bonds between the phosphate-sugar backbone
• staggered cut —> sticky end 
• ligase sticks back the separate DNA fragments
• it stimulates a condensation reaction
• phosphate-sugar backbone binds back to the DNA double helix.
• for this to happen, the same restriction enzymes must be used for the sources. 

this is called recombinant DNA. 

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GENETIC ENGINEERING AND BACTERIA

Why do we carry out genetic engineering?

1. Improving a feature

• plan resistance 
• growth control hormones - myostatic gene promotes muscle growth 

1. Synthesis of useful products

• to produce large quantities for human use (insulin)
• easy access to chemicals - e.g. placing a gene in cows so they produce chemicals in their milk -> pharmaceutical uses 
• insertion of specific genes, - e.g. beta carotene in rice which is a precursor molecules that can be turned into Vitamin A

Bacterial cells and Plasmids in Genetic Engineering

when we identify a gene to be put into another organism - cut with restriction enzymes and then do the same to a plasmid. 

plasmids are separate to the main bacterial chromosomes and carry genes that may show antibiotic resistance - if we mix the plasmids with the gene in ligase enzymes, some may combine to form recombinant plasmids. 

placing the recombinant plasmids back into the bacterial cells 

this can be done by heating and cooling the bacterial cells - from 0-40 degrees and using calcium salts. 

less than 1% will ever take up a plasmid, this is called a transformed bacteria - it is now transgenic

Conjugation

sometimes genetic material can be passed on between bacterial cells and the copies of plasmid DNA are passed between bacteria (ANY) this can be bad because it increases rate at which bacteria become resistant. 

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ENGINEERING CASE STUDIES 

1. HUMAN INSULIN

type I diabetes mellitus: cannot produce insulin

• insulin is a polypeptide (51 amino acids)
• making it hard to find in a genome of 300 million bases (the mapping of genomes for a human were unknown at the time)

so how did they work around this?

Scientist separated out the mRNA of the right length in the pancreatic tissue through centrifugation.

mRNA —————————> DNA

  reverse transcriptase

however not altogether natural so it is only a single complementary strand of DNA. 

• add in free nucleotides and DNA polymerase means a second strand is built using the copied DNA  as a template. 
• this is a copy of the original called the cDNA.
• free nucleotides are added to the end so that they have sticky ends 
• the sticky ends are complementary to the cut plasmid
• plasmid is cut using restriction enzymes. 
• mixed with the cDNA 
• some plasmids take up the gene 
• DNA ligase seal up the plasmids, that are now called recombinant plasmids. (contains new piece of DNA)

1. GOLDEN RICE

Vitamin A comes from Beta-carotene (Pre-cursor molecule)

the part that is eaten : the endosperm has the gene for beta-carotene switched off, third world countries cannot access the vitamin A because of this. 

to active the endosperm cells to generate Beta-cartonene —> enzymes are needed:

• phytoene synthetase —> daffodil bulbs
• Crt 1 enzyme —> soil bacterium

GENE THERAPY

Molecular genetic technology can be used to help cure some genetic disorders. 

-> known as gene therapy

• adding genes (augmentation)

conditions from inhering faulty alleles lead to the loss of a functional gene product (polypepetide) -> engineering the correct genes into the specific specialised cell so that the polypeptide is synthesised and cells can function normally 

• killing specific cells

cause cells to express certain genes to make them more vulnerable

Germline Cell Gene Therapy

insertion of gene during embryonic division