Biotechnology

Advantages of asexual reproduction:

• It is Quick
• It can be completed if sexual reproduction fails or is not possible

All offspring have the same genetic information to enable them to survive in their environment

Disadvantages of asexual reproduction:

• It doesn't produce any genetic variation so any parantal genetic weaknesses will be in all offspring

Vegetative Propagation:Production of structures in an organism thatcan grow into a new individual organism. These offspring are clones of the parent and contain the same genetic information. 

The English Elm reproduces using basal sprots and root suckers, following damage to the parent plant. 

Microproagation by callus tissue culture:

• A samll piece of tissue is taken from the plant (explant)
• The explant is placed on a nutrient growth meduim 
• Cells in the tisue divide, forming a mass of undifferentiated cells called a callus
• After a few weeks, single callus cells can be removed and placed in shoot stimulating hormones
• The growing shoots are transferred onto a different growing medium, with root stimulating hormones
• The growing plants are then transferred to a greenhouse to be acclimatised and grown further

Other Methods of Artificial Vegatative Propagation: 

• Taking cuttings- A section of stem is cut between the nodes, and is treated with plant hormones to encourage root growth
• Grafting- A section of woddy plant is joined to an already growing plant. (Rootstock)

1. Splitting Embryos:

2. Nuclear Transfer- Using enucleated eggs:

• A differentiated cell from an adult can be taken and the nucleus placed inside an egg with its nucleus removed. (An enucleated egg)
  
• They are fussed by Electro-fusion
  
• Then you implant the embryo in surrogate mother uterus. 

Advantages:

• High value animals can be cloned in large numbers
• Rare animals can be cloned to preserve species
• Genetically modified animals can be reproduced quickly

Disadvantages:

• No genetic variation
  
• Unhealthy animals produced by cloning- die earlier
• Not produced with animal welfare in mind

There is the potential of using cloned cells to generate cells, tissues and organs rather than whole organisms. 

Advantages:

• Being genetically identical means no rejection, as the immune system would not recognise them as foreign 
  
• No need to wait for organ donors 
• Cloned cells can be used to generate any type of cell as they are totipotent
• Using cloned cells is less dangerous than a major operation such as a heart transplant 

Possibilities for non-reproductive cloning: 

• Regeneration of heart muscle cells following a heart atack
• Repair of nervous tissue destroyed by diseases such as multiple sclerosis
• Repairing of the spinal cord of those damaged by an accident 

These techniques are Therapeutic cloning. There are ethical objections due to using embryoinic materical and scientific concerns about how cloned cells behave over time 

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

Production of foods:

• Cheese and yoghurt: bacterial growth in milk changes the lfavour and texture of the milk. These bacteria prevent growth of other bacteria that cause food spoliage. 

Production of drugs: 

• Penecillin: The fungus penicillium grown in culture produces the antibiotic as a by product of its metabolism 

Production of enzymes:

• A fungus grown in certain conditions secretes the pectinase enzyme 

The use of bacteria and fungi is widespread because:

• Rapid growth in faveourable conditions, with a generation time of 30 mins
• Often secrete proteins or chemicals into surrounding medium and therefore can be easily harvested 
• Can be genetically engineered to produce specific products 
• Grow well at relatively low temperatures 
• Can be grown anywhere in the world and is not dependant on climate 
• Tend to generate products in a more pure form than those generated by chemical processes
• Can often be grown using nutrient materials that would otherwise be useless or toxic to humans

Lag Phase:

• Organisms are adjusting to their surrounding conditions
• Cells are active but not reproducing so the population remains fairly constant 
• Length of this period depends on growing conditions 

Log Phase:

• The population size doubles each generation as there is enough space and nutrients. 
• Length of this phase depends on how quickly the bacteria reproduce 

Stationary Phase:

• Nutrient levels decrease and waste products rise 
• Individuals die at the smae rate they are being produced 

Death Phase 

• Nutrient exhaustion and waste build up leads to a rapid increase in death rate 

Primary Metabolites:

• Substances produced by an organism as part of its normal growth 
• Includes amino acids, proteins, enzymes, nucleic acids, ethanol and lactate
• Production of primary metabolites matches the growth in population of the organism 

Secondary Metabolites:

• Substances produced by an organism that are not part of its normal growth. 
• Antibiotic chamicals are almost all secondary metabolites
• Production of secondary metabolites usually begin after the main growth period of the organisms and doesn't match the growth in population 

Adsorption: Enzymes are mixed with the immobilising support and bind to it due to hydrophobic and ionic links. Becasue the bonds aren't very strong, enzymes can become detached (leakage). Adsorption can give very high reaction rates 

Covalent Bonding: Enzyme molecules are covalently bonded to a support. This method doesn't immobilise a large quantity of enzymes, but the bodnidng is bery strong so there is little leakage 

Entrapment: Enzymes may be trapped, for example in a gel bed or a network of cellulose fibres. The enzymes will be trapped in their natural state,so their active site will not be affected. However, reaction rates are reduced as the substrate molecules need to get through the trapping barrier

Membrane separation: Enzymes may be physically seperated from the substrate by a partially permeable membrane. substrate and product molecules are small enough to pass through the membrane, and there is no leakage.  

1. Organisms are first mapped to identify which part of the genome they have come from. 

2. Samples of the genome are sheared (Mechanically broken) into smaller sections of around 100000 base pairs. 

3. These sections are placed into seperate bacterial artificial chromosomes (BAC) and transferred to E.Coli cells. as these cells grow, many clones of the sections are reproduced. these cells are reffered to as clone libraries 

4. Cells containing specific BACs are takne and cultured. The DNA is extracted from the cells and a restriction enzyme is used to cut it into maller fragments.

5. The fragments are separated using electrophoresis

6.Is fragment is sequenced using an automated process 

7.Computer programmes then compare overlapping regions from the cuts made by different restriction enzymes in order to reassemble the whole BAC segment sequence

Comparative gene mapping has a wide range of applications:

• The identification of genes found in all or many living organisms gives clues to the relative importance of such genes to life 
• Comparing the DNA of different species shows evolutionary relationships
• Modelling the effects of changes to DNA can be carried out 
• Comparing the genomes from pathogenic and similar but non pathogenic organisms can be used to identify the genes involved in causing disease. This can lead to development of more effective drug treatments
• The DNA of individuals can be analysed. This analysis can reveal mutant alleles or the presence of alleles associated with increased risk of particular diseases, such as heart failure or cancer 

Electrophoresis is used to seperate DNA based on its size. Its is accurate enough so that is can separate fragments that are only different by one base in length. 

The technique uses a gel 'plate' containing agarose which is covered in a buffer solution 

1. DNA samples are treated with restriction enzymes to cut them into smaller fragments

2. The DNA samples are placed into wells at the negative electrode end of the gel

3. The gel is immersed in a tank of buffer solution and an electric current is passed through the solution, ususally for around two hours 

4. DNA is negatively charged due to the many phosphoryl groups. It is attracted towards the psoitive electrode 

5. Shorter lengths of DNA move faster than longer lengths and so move further in the fixed time than the longer pieces of DNA 

6. The position of the fragments can be shown using a dye that stains DNA molecules 

A DNA probe is a short single stranded piece of DNA that is complementary to a section of DNA being investigated. The probe is labeled in one of two ways:

• Using a radioactive marker (Usually by using 32P in the phosphoryl groups) So that the location can be revealed when exposed to photographic film 
• Using a florecent marker that emits a colour on exposure to UV light. 

They will bind to any sample of DNA fragments, where they will bind to any complementary base sequence present. This is called annealing. Probes are usefull in locating specific sequences, for example: 

• To locate a desired gene that is wanted for genetic engineering 
• To identify the same gene on a variety of different geneomes from sepearate species, when conducting genome comparison studies 
• To identify the presence or absence of an allele for a particular genetic disease 

1. The DNA sample is mixed with a supply of DNA nucleotides and the enzyme DNA polymerase 

2. The mixture is heated to 95 degrees centigrade. This breaks the hydrogen bonds 

3. Primers (Short lengths of DNA about 10-20 bases) are added 

4. The temperature is the reduced to about 55 degrees centigrade, allowing the primers to bind forming small sections of double stranded DNA at either end of the sample 

5. DNA Polymerase is added and binds to the double stranded sections 

6. The temperature is raised to 72 degrees centigrade (The optimum temperature for DNA Polymerase) and the enzyme extends the double stranded sections by adding free nuclotides 

7, When DNA Polymerase reaches the other end of the DNA strand, a new double stranded DNA molecule is formed 

8. The process can be repeated many times so the amount of DNA increses exponentially 

In Genetic engineereing, the following steps are necessary:

1. The required gene is obtained: Using the mRNA of a gene, synthesising it using an automated polynucleotide sequencer, or using a DNA probe and restriction enzymes

2. A copy of the gene is plance into a vector: The gene can be sealed into bacterial plasmid using DNA ligase . Genes may also be sealed into virus genome or yeast cell chromosomes 

3. The vector carries the gene to the recipient cell: The gene, once packaged, may be too large to fit in through the cell membrane. There are many different methods used:

• Electroportation-High voltage pulse is applied to distrup the membrane 
• Microinjection-DNA is injected using a very fine micropipette into the host cell nucleus 
• Viral transfer-The vector is a virus
• Ti plasmids- used as vectors can be inserted into a soil bacterium Agrobacterium tumefaciens. This is for use in plants 
• Liposomes- DNA wrapped in lipid molecules which can cross the membrane by diffusion 

4. The recipient cell expresses the gene through protein synthesis 

1. The original gene in people who have normal insulin production will go through transcription in pancreatic cells to give the mRNA. 

2.Once the mRNA is found, the enzyme reverse transcriptase is used to synthesis a complementary DNA strand. 

3.Adding DNA polymerase and a supply of nucleotides to these single strands produces cDNA strands which are complementary to the gene for human insulin.

4.Unpaired nucleotides are added to each end to give sticky ends complimentary to those on the cut plasmid.

5.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 enzyme then seals the recombinant plasmids. 

6.These plasmids are then mixed with bacteria, some of which take up the recombinat plasmid. 

7.The bacteria are then grown on an agar plate, where bacterial cells grow to produce a mound of cloned cells. 

In order to identify the transformed bacteria we use replica plating:

1. The original plasmids contain genes that make any bacteria carrying them ressistant to two different antibiotic chemicals.These resistance genes are known as genetic markers 

2. The plasmids are cut by a restriction enzyme that has its restriction site in the middle of one of the resistance genes, so that if the required gene is taken up, the gene for the resistance dosent work 

3. The bacteria are grown on standard nutrient agar, so all bacterial cells grown form colonies. 

4. Some cells from the colonies are transferred onto agar that contains tetracycline so only those that have not taken up a plasmid will grow

5. Some cells are transferred onto agar containing ampicilin, and only bacteria with the plasmid will grow

6. By keeping track of which colonies are which we know that any bacteria that grew on the first plate have not got the plasmid, but those that grew on the second plate contain the plasmid. 

Rice plants contain the genes that code for the production of beta carotene. However, it is not produced in the endosperm of the rice plants. 

The metabolic process for synthesising beta carotene is complicated, but most of the enzymes needed are already present in the endosperm. It was found that two genes needed to be inserted into the rice plant endosperm to activate the metabolic pathway. 

1. Phytone synthanase is taken from daffodils and is what converts a variety of precursor molecules into phytone 

2. Crt 1 enzyme from bacteria is added, which converts phytone into lycopene (The precursor molecule for the carotnoids) 

3. Enzymes already present in the rice endosperm conver the lycopene into a range of carotenoid molecules

Critics beileive that it will: 1.Lead to a reduction in biodiversity 2.potentially be harmful for humans as the food saftey of enginnered rice is unknown 3. Could contaminate wild rice populations

Gene therapy by augmentation:

• Some conditions are caused by the inheritance of faulty alleles. Engineering a functioning copy of the gene into the relavent specialised cells means that the polypeptide is sythesised 

Gene therapy by killing specific cells:

• Cancers can be treated by eliminating certian populations of cells
• Using genetic techniques to make cancerous cells express genes to produce proteins that make them more vulnerable to attack by the immune system. 

Issues:

• Introduction into somatic cells means that the treatment is short lived 
• Techniques are needed to get the alleles into the specific cells- They may need to be removed
• Genetic manipulations are restricted to the actual patient- wont be passed on to offspring
• Difficulties involved in getting the allele into the genome in a functioning state 

Engineering a gene into the egg or sperm means that as an organism grows, all of its cells will contain a copy of the engineered gene. The functioning allele may be passed on to offspring.

Germline therapy is illegal in humans, as there are many eithical and scientific objections such as:

• An inadvertant modification of the DNA introduced into the germline could create a new human disease or interfere with human evolution. 
• Permanent modifications to the human genome in this way creates moral and ethical issues 

Issues and benefits:

• The functioning allele of the gene is introduced into germline cells- Delivery techniques are more straighforward 
• Introduction into germline cells means that all cells derived from these germline cells contain a copy of the functioning allele. Offspring will have functioning allele 
• Considered unethical to engineer human embryos 

Xenotransplantaion is the transplantation of cell tissues or organs between animals of different species. 

Problems with xenotransplantation:

• Immune rejection issues 
• differences in sizes of organs 
• lifespan of animal used is different to that of a human, so xenograft may age prematurely 
• The body temeprature of different animals is different to ours 
• Animal welfare groups oppose 
• Religious beliefs prevent jewish and muslims eating pork as it is considered dirty 
• Medical concerns about the transfer of disease between animals and humans 

Mircroorganisms:

• Genetically engineered microoragnisms produce useful products 
• Engineered organisms may escape containment and transfer genes to other pathogenic microorganisms . Could lead to widespread antibiotic resistance 

Plants:

• Resistance to pests increases yield, resistance to pesticides allows application and therefore increase yield, accumulation of beta carotene could prevent vit.A deificiency
  
• Genes introduced could pass on to wild relatives. Could result in less genetic variation 
• Genes for herbicide resistance could pass onto unwanted species, giving them resistance
• Modified plants could be toxic to other organisms, or give an allergic response in humans 
• Plants resistant to pathogens could stimulate the more rapid evolution of attack mechanisms in these pathogens 

Animals:

• Pharmaceutical chemicals can be produced in milk 
• Increased milk or meat production
• Production of compatiable organs for transplantation to humans 
• Animal welfare issues arise from genetic manipulation that might lead to animal suffering 
• Strong views about specific animals are held in some religions

Humans 

• Gene threrapies help to treat some genetic dissorders 
  
• The effects of gene transfer are unpredictable 
• Individuals in the germline gene therapy have no say in wether their genetic material should be modified 
• Germline therapy could be used to enahnce desired characteristics (designer babies)
• Concerns about eugenic uses have been raised