2.2 Biotechnology And Gene Technologies Flashcards
0
Q
What method of asexual reproduction is there in eukaryotes?
A
Mitosis
1
Q
What is a clone?
A
- An exact copy
- Genes, cells or whole organisms that carry identical material because they are derived from the same original DNA
2
Q
How do prokaryotes divide?
A
Binary fission
- Their DNA replicates and the cell divides into 2
- Provided there are no mutations, the 2 resulting cells are genetically identical to each other and to the parent cell
3
Q
What are the advantages of asexual reproduction?
A
- It is quick, allowing organisms to reproduce rapidly and so take advantage of resources in the environment
- It can also 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
4
Q
What are the disadvantages of asexual reproduction?
A
- It does not produce any genetic variety
- This means that any parental weaknesses will be in all the offspring
- If the environment changes then all genetically identical organisms will be equally susceptible
5
Q
What is natural vegetative propagation?
A
Asexual reproduction in plants that takes place naturally
6
Q
Some plants (e.g. Elm) are adapted to reproduce asexually following damage to the parent plant. How does this occur?
A
- New growth, in the form of root suckers, or basal sprouts, appears within 2 months of the destruction of the main trunk
- These suckers grow from meristem tissue in the trunk close to the ground, where the least damage is likely to have occurred
7
Q
What are the advantages of vegetative propagation? (To the elm)
A
- Root suckers help the elm to spread, because they can grow all around the original trunk
- When the tree is stressed or the trunk dies, 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
8
Q
What are the disadvantages of vegetative propagation? (To the elm)
A
Dutch elm disease spread through Europe’s elm in the 20th century, killing the leaves, branches and trunks
- The English elm responds to the destruction of the main trunk by growing root suckers
- However, once the new trees get to about 10cm in diameter, they become infected and die in turn
- Because the new trees are clones of the old one, they do not have any resistance to the fungal attack so they remain just as vulnerable as the old one
- There is no genetic variation within the cloned population, so natural selection cannot occur
9
Q
What are the main methods for artificially propagating plants?
A
Taking cuttings
Grafts
10
Q
How are cuttings taken?
A
- A section of stem is cut between leaf joints
- The cut end of the stem is then treated with plant hormones to encourage root growth and planted
11
Q
How is grafting done?
A
- A shoot section of a woody plant is joined to an already growing root and stem
- The graft grows and is genetically identical to the parent plant, but the rootstock is genetically different
12
Q
What is micropropagation?
A
Artificial propagation using plant tissue culture
13
Q
How is micropropagation done?
A
- A small piece of tissue is taken from the plant to be cloned, usually from the shoot tip. This is called an explant
- The explant is placed on a nutrient growth medium
- Cells in the tissue divide but they do not 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 concs that encourage root growth
- The growing plants are then transferred to a greenhouse to be acclimatised and grown further before they are planted outside
14
Q
What are the advantages of micropropagation?
A
- Farmers know what the crop plant produced will be like because it is cloned from plants with known features such as high yield, taste, colour and disease-resistance
- Farmers’ costs are reduced because all the crop is ready for harvest at the same time
- Faster than selective breeding, because huge numbers of genetically identical plants can be generated from a small number of plants
15
Q
What are the disadvantages of micropropagation?
A
- Reduced genetic variation
- Genetic uniformity (all plants are equally susceptible to any new pest, disease or environmental change)
16
Q
What is the central nervous system?
A
The brain and spinal cord
- Made up of grey matter (billions of non-myelinated nerve cells) and white matter (longer, myelinated axons and dendrons that carry impulses)
17
Q
What does the peripheral nervous system consist of?
A
All of the sensory and motor neurones that are outside the CNS (connecting the receptors and effectors to the CNS)
18
Q
What do somatic motor neurones do?
A
Carry impulses from the CNS to skeletal muscles, which are under voluntary control
19
Q
What do autonomic motor neurones do?
A
Carry impulses from the CNS to cardiac muscle, to smooth muscle in the gut wall and to glands, none of which are under voluntary control
20
Q
What is the autonomic system?
A
- A system that operates (to a large extent) independently of conscious control
- It is responsible for controlling the majority of homeostatic mechanisms
- It is capable of controlling the heightened responses associated with the stress response
21
Q
How does the autonomic nervous system differ from the somatic nervous system?
A
- Most autonomic neurones are non-myelinated whilst most somatic neurones are myelinated
- Autonomic connections to effectors always consist of at least 2 neurones (whereas somatic connections to effectors consist of only 1). The 2 neurones connect at a swelling known as a ganglion
- Autonomic motor neurones occur in 2 types: sympathetic and parasympathetic
22
Q
What is an antagonistic system?
A
A system that opposes the action of another system
23
Q
Compare the sympathetic and parasympathetic subsystems
A
- P: Most active in sleep and relaxation, S: most active in times of stress
- P: The neurones of a pathway are linked at a ganglion within the target tissue, so pre-ganglionic neurones vary considerably in length, S: the neurones of a pathway are linked at a ganglion just outside the spinal cord, so pre-ganglionic neurones are very short
- P: post-ganglionic neurones secrete acetylcholine as the neurotransmitter at the synapse between neurone and effector, S: post-ganglionic neurones secrete noradrenaline at the synapse between neurone and effector
- Effects of action include: P: decreased heart rate, pupil constriction, decreased ventilation rate, sexual arousal. S: increased heart rate, puli dilation, increased ventilation rate, orgasm
24
How are the sympathetic and parasympathetic subsystems antagonistic?
- Under normal, resting conditions, impulses are passing along the neurones of both systems at a relatively low rate so the stimulation of the systems is balanced (and there is no overall change to either)
- Changes to internal conditions leads to an altered balance of stimulation between the 2 systems, so they are no longer working antagonistically, leading to an appropriate response.
25
Explain embryo splitting
Cells from a developing embryo can be separated out with each one then going on to produce a separate, genetically identical organism
- Eggs and sperm are collected
- IVF
- 16-cell embryo is grown in vitro
- The embryo is then split into several separate segments
- These segments are then implanted into surrogate mothers
- Each calf is a clone
26
Explain nuclear transfer
- A differentiated cell from an adult can be taken and its nucleus placed in an egg cell which has had its own nucleus removed
- They are combined by electrofusion
- The cell then goes through the stages of development using genetic information from the nucleus
- The cell was taken from the mammary gland of a ewe, its nucleus transplanted into a cell from a second sheep and then inserted into the uterus of a third sheep, and then a fourth, to develop
27
What are the advantages of cloning animals?
- High-value animals can be cloned in large numbers
- Rare animals can be cloned to preserve the species
- Genetically modified animals can be quickly reproduced
28
What are the disadvantages of cloning animals?
- High-value animals are not necessarily produced with animal welfare in mind
- Excessive genetic uniformity in a species makes it unlikely to be able to cope with, or adapt to, changes in the environment
- It is still unclear whether animals cloned using the nuclear matrix of adult cells will remain healthy in the long term
29
What is non-reproductive cloning?
The use of stem cells in order to generate replacement cells, tissue or organs, which may be used to treat particular diseases or conditions of humans
- Also known as therapeutic cloning
30
What are the advantages to non-reproductive cloning?
- Being genetically identical to the individual's own cells means that they will not be 'rejected' because the immune system won't recognise them as foreign
- Cloning and cell culture techniques could mean an end to the current problems of waiting for donor organs to become available for transplant
- Cloned cells can be used to generate any cell type because they are totipotent
- Using cloned cells is likely to be less dangerous than a major operation
31
What are the concerns with non-reproductive cloning?
- Ethical objections to the use of human embryonic material
| - Concerns about a lack of understanding of how cloned cells will behave over ours
32
Why might a sheep produced by nuclear fusion NOT be identical to the sheep it was cloned from (mother sheep)?
- Although the mother's nucleus replaced the nucleus in the egg cell, the mitochondria have not been replaced
- Mitochondrial DNA will be found in there, so the sheep is not genetically identical
33
Define '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
34
What are the 4 major areas in which biotechnology has applications that affect our lives?
- Healthcare and medical processes (e.g. Production of drugs by microorganisms and gene therapy to treat some genetic disorders)
- Agriculture (e.g. Micropropagation of plants and the development of genetically modified plants)
- Industry (e.g. Genetically modifying organisms to produce enzymes)
- Food science (e.g. Developing foods with improved nutrition or better taste, texture and appearance)
35
What are some examples of uses of biotechnological processes and what organism does each use?
Cheese and yoghurt-making: Lactobacillus (bacteria)
Production of mycoprotein: Fusarium (fungus)
Production of penicillin: Penicillium (fungus)
Production of insulin: E.coli (bacteria)
36
What are the advantages to the use of microorganisms in biotechnology?
- Grow rapidly in favourable conditions, with a generation time (time taken for numbers to double) of as little as 30 minutes
- Often produce proteins or chemicals that are given out into the surrounding medium and can be harvested
- Can be genetically engineered to produce specific products
- Grow well at relatively low temperatures
- Can be grown anywhere in the world and are not dependent on climate
- Tend to generate products that are in a more pure form than those generated via chemical processes
- Can often be grown using nutrient materials that would be otherwise useless or even toxic to humans
37
What are the 4 phases in a standard growth curve?
- Lag phase
- Log (exponential) phase
- Stationary phase
- Decline or death phase
38
What happens in the lag phase?
- Organisms are adjusting to the surrounding conditions
- This may mean taking in water, cell expansion, activating specific genes and synthesising specific enzymes
- The cells were active but not reproducing so population remains fairly constant
- The length of this period depends on the growing conditions
39
What happens in the log 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 organisms reproduce and take up the available nutrients and space
40
What happens in the stationary phase?
- Nutrient levels decrease and waste products build up
| - Individual organisms die at the same rate at which new individuals are produced
41
What happens in the 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 will die in a closed system
42
What is a culture?
A growth of microorganisms
43
What is a pure culture?
A culture with a single species
44
What is a mixed culture?
A culture with a mixture of species
45
What can microorganisms be cultured in or on?
- In a liquid such as nutrient broth
| - On a solid surface such as nutrient agar gel
46
What is fermentation?
The process of culturing any microorganism in order to generate a specific product, either aerobically or anaerobically
47
What happens to the population growth if nutrients were added in the lag phase?
The population growth will remain the same, since this organism will still be acclimatising to the environment
48
What would happen to the population growth if nutrients were added in the stationary phase?
There would be another log phase, so population growth would increase again until a factor limits it! bringing it back to the stationary phase
49
What is metabolism?
The sum total of all of the chemical reactions that go on in an organism
50
What are primary metabolites?
- Substances produced by an organism as part of its normal growth; they include amino acids, proteins and enzymes
- Their production matches the growth in population of the organism
51
What are secondary metabolites?
- Substances produced by an organism that are not part of its normal growth
- Their production usually begins after the main growth period of the organisms and so does not match the growth in population of the organism
52
What is a closed culture?
- An environment where all conditions are fixed and contained
- No new materials are added and no waste products or organisms removed
53
Why does temperature need to be controlled in a fermenter and how is it controlled?
- If it gets too hot the enzymes will be denatured
- If it gets too cool, growth will be slowed
- It is controlled using a water jacket
54
Why does the type and time of addition of nutrient need to be controlled in a fermenter?
- Growth of microorganisms requires a nutrient supply, including sources of carbon, nitrogen and any essential vitamins and minerals
- The timing of nutrient addition can be manipulated, depending on whether the process is designed to produce a primary or secondary metabolite
55
Why does oxygen concentration need to be controlled in a fermenter and how is it controlled?
- Most commercial applications use the growth of organisms under aerobic conditions, so sufficient oxygen must be made available
- A lack of oxygen will lead to the unwanted products of anaerobic respiration and a reduction in growth rate
- Can be controlled by continuously releasing oxygen into the culture via air bubbles
56
Why does pH need to be controlled in a fermenter and how is it controlled?
- Changes in pH within the fermentation tank can reduce the activity of enzymes and so reduce growth rates
- Can be controlled using buffers, or by the addition of acid or alkali
57
Why is a pressure vent required in a fermenter?
It prevents any gas build up
58
Why are blades (impellers) required in a fermenter?
They mix the culture evenly
59
Why is a motor required in a fermenter?
To rotate the blades
60
Why is an air inlet required in a fermenter?
Sterile air provides oxygen in aerobic fermenters
61
Why are electronic probes required in a fermenter?
They measure oxygen, pH and temperature levels
62
Why are all inlets and outlets fitted with filters? (In a fermenter)
To prevent contamination
63
Why is an outlet tap required in a fermenter?
To drain fermenter
64
How does a water jacket work? (On a fermenter)
- It allows circulation of water around the fermenter to regulate temperature
- It cools the fermenter, since respiration releases heat
65
How is a batch culture prepared?
- The microorganism starter population is mixed with a specific quantity of nutrient solution
- It is then allowed to grow for a fixed period of time
- No further nutrient is added during this time
- At the end of the period, the products are removed and the fermentation tank is emptied
66
How is a continuous culture prepared?
- Nutrients are added to the fermentation tank at regular intervals
- Products are removed from the fermentation tank at regular intervals
67
What are the disadvantages of batch culture?
- Growth rate is slower because nutrient level declines with time
- Less efficient than continuous, since fermenter is not in operation all of the time
68
What are the advantages of batch culture?
- Easy to set up and maintain
- If contamination occurs, only one batch is lost
- Very useful for processes involving the production of secondary metabolites
69
What are the advantages of continuous culture?
- Growth rate is higher as nutrients are continuously added to the fermentation tank
- More efficient than batch, since fermenter operates continuously
- Very useful for processes involving the production of primary metabolites
70
What are the disadvantages of continuous culture?
- Set up is more difficult than batch, maintenance of required growing conditions can be difficult to achieve
- If contamination occurs, huge volumes of product may be lost
71
What is asepsis?
The absence of unwanted microorganisms
72
What is an aseptic technique?
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 products that are extracted
73
What may happen if unwanted microorganisms contaminate a nutrient medium?
The 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 microorganisms and their products
74
What does the 'immobilisation of enzymes' refer to?
- Any technique where enzyme molecules are held, separated from the reaction mixture
- Substrate molecules can bind to the enzyme molecules
- The products formed go back into the reaction mixture, leaving the enzyme molecules in place
75
What are the advantages of using immobilised enzymes?
- Enzymes are not present with products so purifications/downstream processing costs are low
- Enzymes are immediately available for reuse (particularly useful for continuous processes)
- Immobilised enzymes are more stable because the immobilising matrix protects the enzyme molecules
76
What are the disadvantages of using immobilised enzymes?
- Immobilisation requires additional time, equipment and materials and so is more expensive to set up
- Immobilised enzymes can be less active because they do not mix freely with substrate
- Any contamination is costly to deal with because the whole system would need to be stopped
77
What is downstream processing?
The extraction of enzyme from the fermentation mixture
78
What are the 4 possible methods for immobilising enzymes?
- Adsorption
- Covalent bonding
- Entrapment
- Membrane separation
79
When immobilising enzymes, how does adsorption work?
Enzyme molecules are mixed with the immobilising support and bind to it due to a combination of hydrophobic interactions and ionic links
80
When immobilising enzymes, how does covalent bonding work?
- Enzyme molecules are covalently bonded to a support
| - Often by covalently linking enzymes together and to an insoluble material, using a cross-linking agent
81
When immobilising enzymes, how does entrapment work?
Enzymes may be trapped, for example in a gel bead or a network of cellulose fibres
82
When immobilising enzymes, how does membrane separation work?
- Enzymes may be physically separated from the the substrate mixture by a partially permeable membrane
- The enzyme solution is held at one side of a membrane whilst substrate solution is passed along the other side
83
What are the advantages to using the adsorption method for immobilising enzymes?
Can give very high reaction rates
84
What are the disadvantages to using the adsorption method for immobilising enzymes?
- The bonding forces are not particularly strong, so enzymes can become detached (leakage)
- The strength of the bonds can change the shape of the active site
85
What are the advantages to using the covalent bonding method for immobilising enzymes?
- The binding is very strong, so there is very little leakage of enzyme from the support
86
What are the disadvantages to using the covalent bonding method for immobilising enzymes?
It does not immobilise a large quantity of enzyme
87
What are the advantages to using the entrapment method for immobilising enzymes?
The enzymes are trapped in their natural state (I.e. Not bound to another molecule so their active site will not be affected)
88
What are the disadvantages to using the entrapment method for immobilising enzymes?
Reaction rates can be reduced because substrate molecules need to get through the trapping layer
89
What are the advantages to using the membrane separation method for immobilising enzymes?
- 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
90
How are DNA fragments separated?
By electrophoresis
- A restriction enzyme cuts a piece of DNA wherever it finds a specific sequence of bases
- The lengths of the pieces are then determined using gel electrophoresis
- This uses electricity to separate DNA fragments by size
- The negatively charged DNA fragments are placed in a well at one end of the gel and move towards the positive terminal
- The shorter the fragments, the further they move through the gel
91
What is genomics?
The study of the whole set of genetic information in the form of the DNA base sequences that occur in the cells of the organisms of a particular species
92
What applications are there for comparative genome mapping?
- The identification of genes for proteins found in all or many living organisms gives clues to the relative importance of such genes to life
- Comparing the DNA/genes of different species shows evolutionary relationships
- Modelling the effects of changes to DNA/genes can be carried out
- Comparing genomes from pathogenic and similar but non-pathogenic organisms can be used to identify the genes or base-pair sequences that are most important in causing disease
- The DNA of individuals can be analysed. This can reveal mutant alleles, or the presence of alleles associated with increased risk of particular diseases
93
What functions can non-coding DNA carry out?
Regulatory functions
94
Why does a genome have to be fragmented before sequencing?
- The sequencing reaction can only operate on a length of DNA of about 750 base pairs
- It would be too long and unmanageable if not broken up
95
How is the genome of an organism sequenced?
- Genomes are first mapped to identify which part of the genome (I.e. Which chromosome or section of chromosome) they have come from. Information that is already known is used
- Samples of the genome are sheared (mechanically broken) into smaller sections of around 100000 base pairs
- These sections are placed into separate bacterial artificial chromosomes (BACs) and transferred to E.coli cells
- As the cells grow in culture, many copies of the sections are produced
- These cells are referred to as clone libraries
96
How is a BAC section sequenced?
- Cells containing specific BACs are taken and cultured
- The DNA is extracted from the cells and restriction enzymes are used to cut it into smaller fragments
- The use of different restriction enzymes on a number of samples gives different fragment types
- The fragments are separated by electrophoresis
- Each fragment is sequenced using an automated process
- Computer programmes then compare overlapping regions from the cuts made by restriction enzymes in order to reassemble the whole BAC segment sequence
97
How can DNA fragments be copied?
In a thermocycler
- The DNA fragment to be copied, DNA polymerase, nucleotides and primers are added to the thermocycler
- The thermocycler heats the mixture to 95°C
- This causes the H bonds between the bases to break, causing the 2 strands to separate
- The mixture is cooled to 55°C
- This causes the primers to join with their complementary base pairs at the end of each DNA strand
- DNA polymerase attaches to the double strands
- The temperature is increased to 72°C
- This is the optimum temperature for DNA polymerase
- It adds complementary nucleotides along each DNA strand
- Once the 2 new strands are completed the process is repeated
- The number of DNA strands increases exponentially
- Each cycle takes around 2 mins to complete
98
What does a polymerase chain reaction do?
- It produces large numbers of copies of DNA fragments
- It artificially replicates DNA
- It can be carried out on tiny samples of DNA
101
What does the polymerase chain reaction rely 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 pair rules
102
How is PCR different to natural DNA replication?
- It can only replicate relatively short sequences of DNA, not entire 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 enzyme separates strands in the natural process
103
What are recombinant organisms?
Organisms that have been genetically engineered, by the insertion of a foreign gene into an organism's genome which results in the expression of a new gene
104
What is another name for recombinant organisms?
Transgenic
105
How are DNA copies produced from mRNA?
- Cells are identified that synthesise large amounts of proteins from the desired gene
- These cells contain large quantities of mRNA
- This mRNA must be isolated, for example, by breaking the cells open and centrifuging the contents
- The enzyme reverse transcriptase and a supply of free nucleotides are added
- This results in the synthesis of a single strand of complementary DNA using the extracted mRNA as a template
- Another enzyme, called ribonuclease H, is then added
- This breaks down the mRNA strand, leaving a single-stranded piece of DNA
- DNA polymerase and DNA nucleotides are added
- A complementary strand of DNA is produced
- The result is a fragment of double-stranded DNA that is a copy of the original gene but without the introns
106
What is another name for restriction enzymes?
Restriction endonucleases
107
How are DNA fragments from different organisms joined together?
- Restriction enzymes are used to cut through DNA at specific points
- A particular restriction enzyme will cut DNA wherever a specific base sequence is found
- In most of the restriction enzymes in use, the enzyme catalyses a hydrolysis reaction which breaks the phosphate-sugar backbones of the DNA double helix in different places
- This gives 'sticky ends'
- The vector DNA is cut using the same restriction enzyme
- This means that both sets of DNA will have the same sticky ends
- When separate fragments of DNA need to be stuck together, DNA ligase is used to catalyse a condensation reaction which joins the phosphate-sugar backbones of the DNA double helix together
- The sticky ends are complementary and allows the bases to pair up and hydrogen bond together
- DNA ligase can then seal the backbone
108
How does automated DNA sequencing work?
- The primer joins at the 3' end of the template strand, allowing DNA polymerase to attach
- DNA polymerase adds free nucleotides according to base-pairing rules so the strand grows
- If a modified nucleotide is added, the polymerase enzyme 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
- In each case, the final added nucleotide is tagged with a specific colour
- As these strands run through the machine (in the same way as DNA strands move in electrophoresis) a laser reads the colour sequence, from the strand with only a single nucleotide added, to the 1 with 2 nucleotides added, then 3, then 4 and so on
- The sequence of colours, and so the sequence of bases, can then be displayed
109
What does the reaction mixture for automated sequencing contain?
- The enzyme DNA polymerase
- Many copies of the single-stranded template DNA fragment
- Free DNA nucleotides (some are modified so carry a fluorescent marker)
- Primers
110
What are primers?
- Short, single-stranded sequences of DNA
- They are around 10-20 bases in length
- They are used in sequencing reactions and polymerase chain reactions
- They bind to a section of DNA because the DNA polymerase enzymes cannot bind directly to single-stranded DNA fragments
111
Why is the DNA polymerase enzyme used in PCR described as thermophilic?
It is not denatured by the extreme processes used in the process
112
What are the steps required for genetic engineering?
- The required gene is obtained (by synthesising a cDNA gene from an mRNA strand)
- A copy of the gene is placed (packaged and stabilised) in a vector (using DNA ligase, the gene is inserted into a plasmid)
- The vector carries the gene to the recipient cell
- The recipient expresses the gene through protein synthesis
113
How is a gene placed in a vector?
The gene can be sealed into a bacterial plasmid using the enzyme DNA ligase:
- Large quantities of the plasmid are mixed with bacterial cells, some of which will take up the recombinant plasmid
- The addition of calcium salts and 'heat shock', where the temp of the culture is lowered to around freezing, then quickly raised to 40°C, increase the rate at which plasmids are taken up by bacterial cells
- This process is very inefficient (less than 0.25% of bacterial cells take up a plasmid)
- The bacterial cells that take up a plasmid are known as transformed bacteria
114
What are the 3 types of colony that may grow after bacterial cells have 'taken up plasmids'?
- Bacteria that did not take up a plasmid
- Bacteria that have taken up a plasmid (transformed bacteria)
- Bacteria that have taken up a plasmid that has not sealed in a copy of the gene (it has sealed up on itself to reform the original plasmid)
115
Why is genetic engineering used?
- To improve a feature of the recipient organism
| - To synthesise useful products
116
How can a vector enter the recipient cell?
- Electroporation (a high-voltage pulse is applied to disrupt the membrane)
- Microinjection (DNA is injected using a very fine micropipette into the host cell nucleus)
- Viral transfer (the vector is a virus; this method uses the virus's mechanism for injecting cells by inserting DNA directly)
- Ti plasmids (used as vectors, can be inserted into the soil bacterium Agrobacterium tumefaciens. Plants can be infected with the bacterium, which inserts the plasmid DNA into the plant's genome)
- Liposomes (DNA is wrapped in lipid molecules. These are fat-soluble and can cross the lipid membrane by diffusion)
117
What is bacterial conjugation?
Copies of plasmid DNA are passed between bacteria
118
Why is bacterial conjugation advantageous to bacteria?
It may contribute to genetic variation and survival in the presence of chemicals (antibiotic resistance)
119
What happens in bacterial conjugation?
- 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 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
120
How can transformed bacteria be identified?
Using plasmid vectors with genetic markers
- The original plasmids are chosen because they carry genes that make any bacteria receiving them resistant to 2 different antibiotic chemicals (usually ampicillin and tetracycline)
- The resistance genes are known as genetic markers
- The plasmids are cut by a restriction enzyme that has its target site in the middle of the tetracycline resistance gene, so that if the required gene is taken up, then the tetracycline resistance gene is broken up and will not work
- The gene for ampicillin resistance will still work
A process of replica plating is then used
121
How does replica plating work?
- The bacteria are grown on standard nutrient agar, so all bacterial cells grow to form colonies
- Some cells from the colonies are transferred onto agar that gas 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 has been made with tetracycline so only those that have taken up a plasmid that does not have the insulin gene will grow
- By keeping track of which colonies are which, we know that 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
- These bacteria then produce insulin on a large scale which can be harvested for use
122
What can a lack of vitamin A cause?
Irreversible blindness
123
How do we get vitamin A?
- In the diet, only from animal sources
| - Also derived from the intake of beta-carotene (known as a precursor) which is converted to active vitamin A in the gut
124
What is Golden Rice?
- Rice plants contain the genes that code for the production of beta-carotene
- This molecule is a photosynthetic pigment molecule so is required in the green parts of the plant
- But in the endosperm (the part of the plant that is eaten) the genes for beta-carotene production
- Most of the enzymes for synthesising beta-carotene are already present in the endosperm
- The insertion of 2 genes into the rice genome activates the metabolic pathway in the endosperm cells
- This produces beta-carotene
125
What are arguments against genetically engineered crops?
- It will lead to a reduction in biodiversity
- The human food safety of engineered rice is unknown
- The genetically modified rice could breed with wild types and contaminate wild rice populations
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What is gene therapy?
The treatment of genetic disorders using the techniques of molecular genetic technology
- The potential treatment for genetic diseases, altering the genotype
- The cause of the disease is targeted, rather than just the symptoms
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What is somatic cell gene therapy?
- Involves altering the body cells
- Every cell in the body contains the defective allele, but only the targeted area is affected
- Can be done by adding genes (augmentation)
- Or by killing specific genes
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What is germline cell gene therapy?
- Involves altering sperm or egg cells before fertilisation
- Every cell in the resulting offspring will function normally
- This method is controversial and has never been used on humans, but some studies have been conducted on mice
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How can liposomes be used in somatic cell gene therapy?
- Liposomes are minute lipid spheres which can fuse with the phospholipid bilayer of a cell membrane to release their contents into the cell
- Recombinant DNA technology is used to insert the healthy gene into a plasmid, which is then wrapped in a liposome
- The gene is then released into the cell and moves into the nucleus, where it is expressed
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How are retroviruses and adenoviruses used as vectors?
The genetic material is removed and the new gene is inserted into the viral coat
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What are some of the issues surrounding somatic cell gene therapy
- Genetic manipulations are restricted to the actual patient
- There are difficulties in getting the allele into the genome in a functioning state.
- Introduction into somatic cells means that any treatment is short-lived and has to be repeated regularly. The specialised cells containing the gene will not divide to pass on the allele
- The functioning allele of the gene is introduced into target cells, so techniques to get the gene to the target location are needed or specific cells must be removed, treated and then replaced
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What are some issues concerning germline cell gene therapy?
- Genetic manipulations could be passed on to the patient's children
- Considered unethical to engineer human embryos although more straightforward
- Introduction into germline cells means that all cells derived from these germline cells will contain a copy of the functioning allele
- The functioning allele of the gene is introduced into germline cells - delivery techniques are more straightforward
- Not possible to know whether the allele has been successfully introduced without any unintentional changes to it, which may damage the embryo
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What is xenotransplantation?
Transplantation of cell tissues or organs between animals of different species
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What is allotransplantation?
Transplantation between animals of the same species
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What is a major issue with organ transplants?
Immune rejection of the transplanted tissue
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What problems are associated with using pig organs for transplant to humans?
- Immune rejection
- Differences in organ size
- The body temp of pigs is 39°C (2°C above the average human body temp)
- The lifespan of most pigs is roughly 15 years, so a xenograft may age prematurely
- Some animal welfare groups strongly oppose killing animals in order to harvest their organs for human use
- Religious beliefs (Orthodox Jewish + Muslim faiths prohibit eating pork)
- Medical concerns exist about possible disease transfer between animals and humans
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What are some advantages to genetic manipulation?
- Genetically engineered microorganisms produce useful products
- Could be used to prevent vitamin deficiencies
- Resistance to pesticides allows application of weedkillers and increase in yield
- Resistance to pests increases yield
- Pharmaceutical chemicals can be produced in milk
- Increased milk or meat production
- Production of compatible organs for transplantation to humans
- Gene therapies treat some genetic disorders
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What are some disadvantages to genetic manipulation?
- Engineered microorganisms may escape from containment and transfer genes to other, pathogenic microorganisms
- Genetic engineering often uses antibiotic resistance genes as markers. These genes could be passed to other microorganisms, leading to more widespread antibiotic resistance
- Genes introduced to crop plants may pass to wild relatives it is thought that this could result in less genetic variation and/or the production of less useful hybrid crops
- Genes could pass to weed/unwanted species giving them herbicide or pesticide resistance
- Genes for pest resistance could pass to other plant species, changing the stability of biological communities and possibly affecting many other organisms and food chains
- Modified plants may be toxic to other organisms, or lead to allergic responses in humans
- Plants resistant to pathogens could stimulate the more rapid evolution of attack mechanisms in these pathogens
- Animal welfare issues arise from genetic manipulations that might lead to animal suffering
- Strong views about specific animals are held in some religions
- The effects of gene transfer are unpredictable
- Individuals resulting from germline cell gene therapy would have no say in whether their genetic material should have been modified
- There are concerns that germline cell gene therapy could be used not only to eliminate disease, but also to enhance favourable characteristics
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What are DNA probes?
A DNA probe is a short single-stranded piece of DNA that is complementary to a section of the DNA being investigated
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How can a DNA probe be labelled?
- 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
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When are probes useful?
In locating specific sequences:
- 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, when conducting genome comparison studies
- To identify the presence or absence of an allele for a particular genetic disease
