Biotechnology and gene technologies

Advantages

• It is quick - Allows plants to reproduce rapidly and take advantage of resources in the enviroment.
• Can be completed if sexual reproduction fails or isn't possible.
• All offspring will have the genetic information to enable them to survive in their evnviroment.

Disadvantages

• Doesn't produce any genetic variety - Any genetic parental weakness will be present in all the offsping making all genetically identical organisms susceptible to the same enviromental changes.
  

Vegetative propagation is asexual reproduction in plants making use of specialised vegetative structures that grow to form new and separate individual species.

A number of plant species are adapted to reprouce asexually following damage to the parent plant, this includes the English elm. This allows the species to survive catastrophes such as disease or burning.

New growths in the form of root suckers or basal sprouts, appear within 2 months of the destruction of the main trunk. The suckers grow from meristem tissue in the trunk close to the ground, where the least damage is likely to have occured.

When the tree is stressed or the trunk dies (when the tree is felled as part of the coppice cycle) the suckers grow into a circle of new elms called a clonal patch. This, in turn puts out new suckers so that the patch keeps expanding as far as resources permit.

In the 20th century Dutch elm disease spread through Europe's elms, this is a fungal disease carried by a beetle. As a result the leaves withered, follwed by death of the branches and trunks. In response to this disease destroying the main trunk, the English elm grew root suckers, however once they reached 10cm in diameter they became infected and died in turn.

This is due to the new trunks being clones of the old one, they do not have any resistance to the fungal attack, making them just as vunerable as the original tree. There is no genetic variation within the cloned populatin, so natural selection can't occur.

Two main methods are:

• Taking cuttings - A section of the stem is cut between leaf joints (nodes). The cut end of the stem is then treated with plant hormones to encourage root growth, and planted. The cutting forms a new plant that is a clone of the original parent plant.
• Grafting - A shoot section of a woody plant is joined to an already growing root and stem (rootstock). The graft grows and is genetically identical to the parent plant, but the rootstock is gentically different.

Tissue culture also called micropropagation is the cloning of isolated cells or small pieces of plant tissues in special culture solutions, under controlled aseptic conditions.

Tissues cultures can be used to generate large stocks of a particularly valuable plant very quickly. These stocks are also known to be disease-free.

Micropropagation by callus tissue culture

• An explant (small piece of tissue) is taken from the plant to be cloned.
  
• The explant is placed in a nutrient growth medium.
• Cells in the tissue divide, but they don't differentiate. Instead they form a mass of undifferentiated cells called a callus.
  
• After a few weeks, single callus cells can be removed from the mass and placed on a growing medium containing plant hormones that encourage shoot growth.
• After a further few weeks, the growing shoots are transferred onto a different growing medium containing different hormone concentrations that encourage root growth.
• The growing plants are then transferred to a greenhouse to be acclimatised and grown futher before they are planted.

Advantages:

• Much faster than selective breeding.
• Huge numbers of gentically identical plants can be generated from a small number of plants or a single valuable plant.
• Farmers know what the crop plant produced will be like becuse it is a clone.
• Farmers' costs are reduced because all the crop is ready for harvest at the same time.

Disadvantages:

• Genetic uniformity means that all plants are equally susceptible to any new pest, disease or enviromental change.

• The eggs of a high-value female and the sprem of a high-value male are collected.
• In vitro fertilisation takes place.
• The embryo is grown in virto until it is 16 cells.
• The embryo is then split into several seperate segments.
• Each segment is implanted into surrogate mothers.
• Each baby produced is a clone.

• A differentiated cell from an adult is taken along with an ovum cell from another adult which has had it's nucleus remove,this is called a enucleate ovum.
• Electro-fusion then occurs.
• The cell is then reconstructed with the cytoplasm of the adut that donated the ovum and the nuclus of the differetiated cell.
• The cell is then cultured in the tied oviduct of another adult.
• The early embryo is recovered and implanted in a surrogate mother's uterus.
• A clone of the adult that donated the differentiated cell is born.

Advantages:

• High-value animals can be cloned in large numbers. E.g. Cows giving high milk yield.
• Rare animals can be cloned to preserve the species.
• Genetically modified animal can be quickly reproduced. E.g. Sheep that produce pharmaceutical chemicals in their milk.

Disadvantages:

• High-value animals aren't necessarily produced with animal welfare in mind. E.g. Some strains of meat-producing chickens have been developed that are unable to walk.
• Excessive genetic uniformity in a species makes it unlikelt to be able to cope with, or adapt to, changes in the enviroment.
• It is unclear whether animals cloned using the nuclear materials of adult cells will remain healthy in the long term.

Non-reproductive cloning also know as therapeutic cloning is the use of stem cells in order to generate replacement cells, tissues or organs, which may be used to treat particular diseases or conditions of humans.

Advantages:

• Being gentically identical to the individual's own cells means they won't be rejected by the immune system as it won't recognise them as foreign.
• Could mean an end to the problems of waiting for donor organs to become avalible for transplants.
• Cloned cells can be used to generate any cell type because they are totipotent.
• Using cloned cells us likely to be less dangerous than a mjor operation such as a transplant.

Possiblities:

• Regeneration of heat muscle cells following a heart attack.
  
• Repair of nervous tissue destroyed by dieases. E.g. Multiple sclerosis
• Repairing the spinal cord of those paralysed by an accident.

Biotechnology is the use of microorganisms or biochemical reactions to generate useful products.

Purpose of biotechnological process:

• The production of foods. - Cheese and yoghurt making using Lactobacillus.
  
• The production of drugs or other pharmaceutical chemicals. - Penicillin using Penicillium.
• The production of enzymes or other chemicals for commercial use. - Pectinase, used in fruit juice extraction produced by A. niger.
• The bioremediation of waste products. Waste water treatment

A small number of organisms placed in a fresh 'closed culture' enviroment will undergo population growth in a very predictable, standard way.

• Lag phase - Organisims are adjusting to the surrounding conditions (taking in water, cell expansion, activating specific genes and synthesising specific enzymes) the cells are active but not reproducing so population remains fairly constant. The length of this period depends on growing conditions.
• Log (exponential) phase - The population size doubles each generation as every individual has enough space and nutrients to reproduce. The length of this phase depends on how quickly the organism reproduce and take up the avalible nutrients and space.
• Stationary phase - Nutrient levels decrease and waste products like carbon dioxide and other metabolites build up. Individual organisms die at the same rate at which new individuals are being produced. (In an open system this is the carrying capacity of the enviroment)
• Decline or death phase - Nutrient exhaustion and increased levels of toxic waste products and metabolites lead to the death rate increasing above the reproduction rate. Eventually all organisms in a closed system will die.
  

Primary metabolites are any metabolites formed as part of the normal growth of a microorganism.

They include amino acids, proteins, enzymes, nucleic acids, ethanol and lactate.

The production of primary metabolites matches the growth in population of the organism.

Secondary metabolites are any metabolites produced by a microorganism that are not part of its normal growth.

Usually anitibiotic chemicals.

Production usually begins after the main growth period of the microorganism so it doesn't match the growth in population of the organism.

Whilst all microorganisms produce primary metabolites only a small number of microorganisms produce secondary metabolites.

The growing conditions in a fermenter can be manipulated and controlled in order to ensure the best possible yeild of the product. The growing conditions depend on the microorganism being cultured and on whether the process is designed to produce a primary or secondary metabolite. They are:

• Temperature - too hot and enzymes will denature, too cold and growth will be slowed.
• Type and time of addition of nutrient  
• Oxygen concentration - a lack of oxygen will lead to the unwanted products of anaerobic respiration and reduction in growth rate.
• pH - changes in pH can reduce the activity or enzymes and so reduce growth rates.

Such large cultures require 'starter' populations of the microorganism, this are obtained by taking a pure culture and growing it in sterile nutrient broth.

A batch culture is a culture of microorganisms that takes place in a single fermentation. Products are separated from the mixture at the end of the fermentation process.

• Growth rate is slower because nutrient levels decline with time.
• Easy to set up and maintain.
• If contamination occurs only one batch is lost.
• Less efficeint, fermenter is not in operation all of the time.
• Very useful for processes involving the production of secondary metabolites.

Penicillin is produced using batch culture of Penicillium fungus.

A continuous culture is a culture of microorganisms set up in a reaction vessel to which substrates are added and from which products are removed as the fermentation process continues.

• Growth rate is higher as nutrients are continuously added to the fermentation tank.
• Set up is more difficult, maintenance of required growing conditions can be difficult to achieve.
• If contamination occurs, huge volumes of products may be lost.
• More efficient, fementer operates continuously.
• Very useful for processes involving the production of primary metabolites.

Insulin is produced from continuous culture of geneticall modified E.coil bacteria.

Asepsis is the absence of unwanted microorganisms.

Aseptic technique refers to any measure taken at any point in a biotechnological process to ensure that unwanted microorganisms do not contaminate the culture that is being grown or the prodcuts that are extracted.

Unwanted microorganisms:

• Compete with the culture microorganisms for nutrients and space.
• Reduce the yield of useful products from the culture microorganisms.
• May cause spoilage of the product.
• May produce toxic chemicals.
• May destroy the culture microorganism and their products.

In large-scale culture levels a number of different measures are used to ensure aseptic technique:

• Washing, disinfecting and steam-cleaning the fermenter and pipes when not in use removes excess nutrient medium and kills microorganisms.
• Fermenter surfaces made of polished stainless steel prevent microbes and medium sticking to surfaces.
• Sterilising all nutirent media before adding to the fermenter prevents introduction of contaminants.
• Fine filters on inlet and outlet pipes avoid microorganism entering or leaving the fermentation vessel.

Immobilisation of enzymes refers to techniques where enzyme molecules are held separated from the reaction mixture. Substrate molecules can bind to the enzyme molecules and the products formed go back into the reaction mixture leaving the enzyme molecules in place. 

Advantages:

• Enzymes aren't present with products so purification costs are low.
• Enzymes are immediately available for reuse allowing a continuous process.
• Immobalised enzymes are more stable because the immobilising matrix protects the enzyme molecules.

Disadvantages:

• Immobilisation requires additional time, equpiment and materials and so is more expensive to set up.
• Immobalisied enzymes can be less active because they don't mix freely with substrate.
• Any contamination is costly to deal with because the whole system would need to be stopped.

Adsorption

Enzyme molecules are mixed with the immbolising support and bind to it due to a combination of hydrophobic interactions and ionic links. Because the bonding forces aren't particularly strong, enzymes can become detached (known as leakage). However provided the enzyme molecules are held so that their active site is not changed and is displayed, adsorption can give very high reaction rates.

Adsorbing agents used include porous carbon, glass beads, clays and resisns.

Covalent bonding

Enzyme molecules are covalently bonded to a support, often by convalently linking enzymes together and to an insoluble material (clay particles) using a cross-linking agent (gluterakdehyde or sepharose). The method doesn't immboilise a large quantity of enzymes but binding is very strong so there is very little leakage or enzymes from the support.

Entrapment

Enzymes may be trapped, for example in a gel bead or a network of cellulous fibres. The enzymes are trapped in their natural state. However, reaction rates can be reduced because substrate molecules need to get through the trapping barrier, this means the active sites are less easily avalible than with adsorbed or covalently bonded enzymes.

Membrane separation

Enzymes may be physically separated from the substrate mixture by a partially permeable membrane. The enzyme solution is held at one side of the membrane whilst substrate solution is passed along the other side. Substrate molecules are small enough to pass through the membrane so that the reaction can take place. Product molecules are small enough to pass back through the membrane.

Electrophoresis is a method used to separate molecule in a mixture based on their size. The substances within the mixture must have a charge. When a current is applied, charged molecules are attracted to the oppositely charged electrode. The smallest molecules travel fastest through the stationary phase and will travel the furthest, so the molecules are separated by size.

The technique uses a gel slab containg argarose which is covered in buffer solution. Electrodes are attached to the end of the gel so that a current can be passed through it.

• DNA samples are treated with restriction enzymes to cut them into fragments.
• The DNA samples are placed into wells cut into the negative electrode end of the gel.
• The gel is immersed in a tank of buffer solution and an electric current is passed throug hthe solution for a fixed period of time, usually around 2 hours.
• DNA is negatively charged because of the many phosphoryl groups. It is attracted to the positive electrode, so the DNA fragments diffuse through the gel and towards the positive electrode end.
• Shorter lengths of DNA move faster than longer lengths and so move further in the fixed time that the current is passed through the gel.
• The position of the fragments can be shown using a dye that stains DNA molecules.
• A nylon or nitrocellulose sheet is placed over the gel, covered in paper towel, pressed and left overnight (blotting).
• The DNA fragments are transferred to the sheet and can now be analysed.
• The DNA fragments can be made visible by labelling the DNA with radioactive markers before the sample is run. Placing photographic film over the sheet shows the position of DNA samples in finished gel.

A DNA probe is a short single-stranded piece of DNA (about 50-80 nucleotides long) that is complementary to a section of DNA being investigated.

The probe is labelled one of two ways:

• Using a radioactive marker so that the location can be revealed by exposure to photographic film.
• Using a fluorescent marker that emits a colour on exposure to UV light. Fluorescent marked are also used in automated DNA sequencing.

Copies of the probe can be added to any sample of DNA fragments, due to them being single stranded, they will bind to any fragment where a complementary base sequence is presenct, this is known as annealing.

Probes are useful in locating specific sequences, for example:

• To locate a specific desired gene that is wanted for genetic engineering.
• To identify the same gene on a variety of different genomes, from separate species.
• To identify the presence or absence of an allele for a particular genetic disease.

PCR is artificial DNA replication. The sequencing reaction relies on the fact that DNA:

• Is made up of antiparallel backbone strands.
• Is made of strands that have a 5' (prime) end and a 3' (prime) end.
• Grows only from the 3' end.
• Base pairs pair up according to complementary base-pairing rules.

PCR is not identical to natural DNA replication:

• It can only replicate relatively short sequences of DNA, not entrie chromosomes.
• The addition of primer molecules is required in order for the process to start.
  
• A cycle of heating and cooling is used in PCR to separate and bind strands; DNA helicase enzymes separate strands in the natural process.

Primers are short, single-stranded squences of DNA, around 10-20 bases in length. They are needed, in squencing reactions and polymerase chain reactions, to bind to a section of DNA because the DNA polymerase enzymes cannot bind directly to single-stranded DNA fragments.

• The DNA sample is mixed with a supply of DNA nucleotides and the enzyme DNA polymerase.
• The mixture is heated to 95oC. This breaks the hydrogen bonds holeding the complementary strands together, making the samples single-stranded.
• Primers are added.
• The temperature is reduced to around 55oC, allowing the primers to bind (hydrogen bonding) and form small sections of double-stranded DNA at either end of the sample.
• The DNA polymerase can bind to these double stranded sections.
• The temperature is raised to 72oC (the optimum temperature for DNA polymerase). DNA polymerase extends the double-stranded section by adding free nucleotides to the unwound DNA. (in the same way as nautural DNA replication).
• When DNA polymerase reaches the other end of the DNA strand, a new double-stranded DNA molecule is generated.
• The whole process can be repeated many times so the amount of DNA increases exponentially.

DNA polymerase is described as thermophilic because it isn't denatured by the extreme temperatures used in the process. The enzyme is derived from Thermus aquaticus which grows in hot springs.

The reaction mixture contains:

• DNA polymerase.
• Many copies of the single-stranded template DNA fragment.
• Primers.
• Free DNA nucleotide. Some of the nucleotides carry  fluorescent marker, these nucleotides are modified and if they are added to the growing chain the DNA polymerase is 'thrown off' and the strand can't have any further nucleotides added. Each nucleotide has a different coloured fluorescent marker.

The reaction proceeds a followed:

• The primer anneals at 3' end of the template strand, allowing DNA polymerase to attach.
• DNA polymerase adds free nucleotides according to base-pairing rules sothe strand grows.
• If a modified nucleotide is added, the polymerase enxyme is thrown off and the reaction stops on that template strand.
• As the reaction proceeds many molecules of DNA are made, the fragments generated vary in size. The final nucleotide added is tagged with a specific colour.
• As these strands run through the machine a laser reads the colour sequence from the strands in order from the strand with the lowest amount of nucleotides to the one with the highest amount of nucleotides. The sequence of colours, and so the squence of bases can then be displayed.

Restriction enzymes cut through DNA at specific points. A particular restricition enzyme will cut DNA wherever a specific base sequence occurs and only where that sequence occurs. This sequence is called the restriction site. The enzyme catalyses a hydrolysis reaction wich breajs the phosphate-sugar backbones of the double helix in different places and leave exposed bases know as sticky ends.

A sticky end is formed when DNA is cut using a restriction enzyme. It is a short run if unpaired, exposed bases seen at the end of the cut section.

DNA ligase is used to catalyse a condensation reaction which joins the phosphate-sugar backbones of the DNA double helix together. In order to join DNA fragments from different sources both need to have originally been cut by the dame restiriction enzyme, this means that the sticky ends are complementary and allows the bases to pair up and hydrogen bond together.

When DNA fragments from different organisms are joined in this way the resulting DNA is called recombinant DNA.

There are 2 main reasons for carrying out genetic engineering:

1. Improving a feature of the recipient organism.

• Inserting a gene into a crop plant to give the plant resistant to herbicides allows farmers to use herbicides as the crops are growing and increase crop yeild.
• Inserting a growth-controlling gene into livestock to promote muscle growth.

2. Engineering organisms that can sysnthesise useful products.

• Inserting the gene for insulin or growth hormone, into bacteria and growing the bacteria produces large quantities of the hormone for human use.
• Inserting the gene for a pharmaceutical chemical into a female sheep so that the chemical is produced in her milk means the chemical can be easily collected.
• Inserting genes for beta-carotene production into rice so that the molecule is present in the edible part of the rice plant.

• The gene that has been identified to be placed in another organism can be cut from DNA using a restriction enzyme and then placed into a plasmid (small circular piece of DNA).
• If the plasmids are cut with the same restriction enzyme as that used to isolate the gene then complementary sticky ends are formed.
• Mixing quantities of plasmid and gene in the presence of ligase enzyme means that some plasmids will combine with the gene, which then becomes sealed into the plasmid to form a recombinant plasmid.
• Many of the cut plasmids in the presence of ligase enzyme will reseal to reform the original plasmid.
• Large quantities of the plasmid are mixed with bacteria cells, some will take up the recombinant plasmid.
• The addition of calcium salts and 'heat shock' (the temperature of the culture is lowered to around freezing, then quickly raised to 40oc) increases the rate at which plasmids are taken up by baterial cells.
• Bacteria that do take up a recombinant plasmid are known as transformed bacteria.
• The transformation results in bacteria containg new DNA making them transgenic.

Conjugation - bacterial cells can join together and pass plasmid DNA from one bacterial cell to another.

Copies of plasmid DNA are passed between bacteria, sometimes even of different species. This swapping of plasmids is of concern because plasmids ofter carry genes associated with resistance to anitbiotics, being able to swap plasmids speeds the spread of antibiotic resistance between bacterial populations.

• A conjugation tube forms between a donor and a recipient. An enzyme makes a nick in the plasmid.
• Plasmid DNA replication starts. The free DNA strand starts moving through the tube.
• In the recipient cell, replication starts on the transferred DNA.
• The cells move apart and the plasmid in each forms a circle.

• mRNA and reverse transcriptase are used to synthesise a complementary DNA strand.
• DNA polymerase and DNA nucleotides are then added to the single strands of DNA. A second strand is built on using the copied DNA as a template.
• A copy of the original gene called a cDNA gene is produced. Unpaired nucleotides are added at each end to the give sticky ends complementary to those on the cut plasmid.
• Plasmids are then cut open with a restriction enzyme and mixed with the cDNA genes. Some of the plasmids take up the gene.
• DNA ligase thaen seals up the plasmids forming recombinant plamids.
• The recombinant plasmids are then mixed with bacteria, some of which take up the recombinant plasmids.
• The bacteria are then grown on an agar plate, where each bacterial cell produces a colony.

There are 3 possible types of colony that may grow:

• Some form bacteria that didn't take up a plasmid.
• Some from bacteria that have taken up a plasmid that hasn't sealed in a copy of the gene but has sealed up on itself to reform the original plasmid.
• Some that have taken up the recombinant plasmid, these are transformed bacteria.

• The originally plasmids carry genes that make any bacteria receiving them resistant to two antibiotic chemical (usually ampicillin and tetracycline). These resistant genes are genetic markers.
• The plasmids are cut by a restriction enzyme that has its target site in the middle of the tetracylcine resistance gene, so that if the required gene is taken up, then the gene for tetracyline resistance is broken up and doesn't work. But the gene for ampicillin resistance still works.

Replica plating is then used.

• The bacteria are grown on a standard nutrient agar, so all bacterial cells grow to form colonies.
• Some cells form the colonies are transferrred onto agar that has been made with ampicillin so only those that have taken up a plasmid will grow.
• Some cells from these colonies are transferred onto agar that had been made with tetracycline so only those that have taken up a plasmid that doesn't have the insulin gene will grow.
• Any bacteria that grow on the ampicillin agar, but not on the tetracycline agar, must have taken up the plasmid with the insulin gene.
• We can now identify the colonies we want and grow them on a large scale.

Somatic cell gene therapy involves the placing the gene in adult differentiated cells.

• Gene therapy by adding genes (augmentation): Some conditions are caused by the inheritance of faulty alleles leading the the loss of a functional gene product (polypeptide). Engineering a functioning copy of the gene into the relevant specialised cells means that the polypeptide is synthesised and the cells can function normally.
• Gene therapy by killing specific cells: Cancers can be treated by eliminating certain populations of cells. Using genetic techniques to make cancerous cells express genes to produce proteins that make the cells vulnerable to attack by the immune system could lead to targeted cancer treatments.

Issues:

• Introduction into somatic cells means that any treatment is short-lived and has to be repeated regularly. The specialised cells contian the gene won't divied to pass on the allele.
• Genetic manipulations are restricted to the actual patient.
• There are difficulties getting the allele into the genome in a functioning state.
• Techniques to get the gene to the target location are needed, or specific cells must be remoed, treated and tne replaced.

Germline gene therapy involves placing the gene into embryonic cells.

Engineering a gene into sperm, egg, zygote or into all the cells of an early embryo means that as the organism grows, every cell contains a copy of the engineered gene that can then function within any cell where the gene is required.

Issues:

• It is deemed unethical to engineer human embryos.
• It isn't possible to know whether the allele  has been successfully introduced without any unintentional changes to it, which may damage the embryo.
• An inadvertent modification of DNA introduced into the germline could create a new human disease or inerfere with human evolution in an unexpected way.

Positives:

• Delivery techniques are more straightforwards as the allele is introduced into germline cells.
• All cells derived from these germline cells will contain a copy of the functioning allele. The offspring may also contain the allele.