Biotechnology and Gene Technologies

Question 1

What is biotechnology?

Answer 1

The industrial use of living organisms (or parts of living organisms) to produce food, drugs or other products

Question 2

Why are microorganisms often used in biotechnological processes (7 reasons) ?

Answer 2

They grow rapidly in favourable conditions
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 in low cost conditions
Can be grown anywhere in world; not dependent of climate
Tend to generate products that are more pure than those generated by chemical means
Can often be grown on nutrient materials that would otherwise go to waste

Question 3

Describe the standard growth curve of microorganisms in a closed culture

Answer 3

Lag phase: low number; gradient of approx. 1
Log/exponential phase: rapid increase in number of microorganisms
Stationary phase: microorganism reproduction rate equals the death rate; no change in population size
Death phase: population size decreases as death rate exceeds reproduction rate

Question 4

What is the lag phase?

Answer 4

Organisms are adjusting to the surrounding conditions. This may mean taking in water, cell expansion, activating specific genes and synthesising specific enzymes. Cells are active but are not reproducing, so population size is fairly constant. Length of period depends on growing conditions

Question 5

What is the log phase?

Answer 5

Population size doubles each generation as every individual has enough space and nutrients to reproduce. In some bacteria, the population can double every 30 minutes in these conditions. Length of this phase depends on how quickly the organisms reproduce and take up the available nutrients and space.

Question 6

What is the stationary phase?

Answer 6

Nutrient levels decrease and waste products like carbon dioxide build up. Individual organisms die at the same rate at which new individuals are being reproduced. (In an open system, this would be the carrying capacity of the environment)

Question 7

What is the death phase?

Answer 7

Nutrient exhaustion and increased levels of toxic wast products and metabolites lead to the death rate increasing above the reproduction rate. Eventually, all organisms will die in a closed system

Question 8

How can enzymes be immobilised (4 methods)?

Answer 8

Adsoption: enzymes are mixed with the immobilising support and bind to it due to hydrophobic interactions and ionic links. Support could be porous carbon, glass beads, clay and resins
Covalent bonding: enzymes are covalently bonded to a support; often linking enzymes together to an insoluble material
Entrapment: enzymes are trapped in a gel bead/cellulose fibres. Not bound to another molecule
Membrane separation: enzymes are physically separated from the substrate mixture by a partially permeable membrane. Substrate and products can pass through the membrane; enzyme can’t

Question 9

What are the advantages of immobilising enzymes in large scale production?

Answer 9

Enzymes aren’t present with products so purifications/downstream processing costs are cut
Enzymes are immediately available for reuse. This is good for allowing continuous processes
Immobilised enzymes are more stable because the immobilising matrix protects the enzyme molecule

Question 10

Compare batch and continuous cultures

Answer 10

Continuous cultures have higher growth rate as the nutrient levels are constantly replenished and don’t decline over time
Batch cultures are easy to set up and maintain
Contaminations in batch cultures only result in one batch lost; in continuous cultures much product is lost and the whole system has to be sterilised
Batch cultures are less efficient as the fermenter isn’t always operational
Batch cultures are good for production of secondary metabolites; continuous for primary metabolites

Question 11

What is a primary metabolite?

Answer 11

Substances produced by an organism as a part of its normal growth. They include amino acids, proteins, enzymes, nucleic acids, ethanol and lactate. The production of primary metabolites matches the growth in population of the organism

Question 12

What is a secondary metabolite?

Answer 12

Substances produced by an organism that are not a part of its normal growth. They antibiotic chemicals produced by a number of microorganisms are almost all secondary metabolites. The production of secondary metabolites usually begins after the main growth phase of the organism and so does not match the growth in population. Not all microorganisms produce secondary metabolites

Question 13

What is the importance in manipulating the growing conditions in a fermenter?

Answer 13

Want the maximum yield of product, so need to ensure we maintain conditions for best rate and yield. For example:
Temperature must be maintained at optimum for enzymes
Right nutrient must be added at the right time to get primary/secondary metabolite
Oxygen concentration must be plentiful so no anaerobic respiration products and growth rate not limited
pH must be regulated so as not to reduce enzyme activity

Question 14

Why is asepsis important in manipulating microorganisms?

Answer 14

Nutrient medium could also support unwanted microorgansims: contaminants. They compete with culture for nutrients and space; reduce yield of useful products; may cause spoilage of the product; may produce toxic chemicals and may outcompete the culture.

Question 15

What are the differences between reproductive and non-reproductive cloning?

Answer 15

Reproductive cloning produces a new organism at the end; whilst non-reproductive cloning produces a culture of identical cells that have the potential to be used in gene therapy techniques

Question 16

How are natural clones in plants produced?

Answer 16

Root suckers in elm trees
Tubers in potatoes
Bulbs in onions
Runners in strawberries

Question 17

Describe vegetative propagation in elm trees

Answer 17

Following stress to the parent tree, root suckers or basal sprouts appear around the trunk, grown from the meristem tissue in the trunk close to the ground where the least damage is likely to have occurred. Root suckers grow in a circle called a clonal patch that continues expanding as long as resources allow. However, there is no genetic variation in the clonal patch

Question 18

Describe how vegetative propagation can occur artificially

Answer 18

Cuttings: plant is cut between leaf nodes and end placed in medium with plant hormones to encourage root growth. New plant is clone of original plant
Grafting: existing root and stem has a shoot section of a woody plant joined to it, aligning xylem and phloem vessels as much as possible. The shoot is genetically identical, but the roots are different

Question 19

Describe the production of artificial clones of plants from tissue cultures

Answer 19

Micropropagation by callus tissue culture.
Small piece of tissue is taken from plant shoot tip called explant. Explant is placed on nutrient growth medium. Cells divide but don’t differentiate and form a callus. Single callus cells can be removed and placed on a growth medium containing plant hormones that encourage shoot growth. Transferred to medium that encourages root growth. Finally placed in greenhouse to grow more.

Question 20

What are the advantages of plant cloning in agriculture?

Answer 20

Know what crop plant will be like as they are cloned from plants with known features
Crop is ready to harvest all a the same time
Some plants can only be grown from clones as the cultivated fruits are sterile (e.g. bananas)
Micropropagation is much faster than selective breeding, as many identical plants can be made from a single valuable plant

Question 21

What are the disadvantages of plant cloning in agriculture?

Answer 21

Genetic uniformity means all plants are equally susceptible to any new pest, disease or environmental change

Question 22

How can animals be artificially cloned (2 methods)?

Answer 22

Splitting embryos: cells from a developing embryo grown in vitro separated out before 16 cell stage, each one going to produce a genetically identical organism. Cells implanted into a surrogate
Somatic cell nuclear transfer (SCNT): remove nucleus from egg cell (enucleated); take nucleus from somatic cell and inject into the enucleate ovum. Place on growth medium and encourage division with electro-fusion. Implant embryo into surrogate mother, get an organism that has nuclear DNA of somatic cell donor and mitochondrial DNA of the egg cell donor

Question 23

What are the advantages of cloning animals?

Answer 23

High-value animals (e.g. high milk yield) can be cloned in large numbers
Rare animals can be cloned to preserve the species
Genetically modified animals can be quickly reproduced

Question 24

What are the disadvantages of cloning animals?

Answer 24

High-value animals aren’t necessarily produced with animal welfare in mind (some meat producing chickens can’t walk)
Excessive genetic uniformity in a species makes it unlikely to be able to cope with changes in environment
It is unclear whether animals cloned using nuclear material of adult cells will remain healthy in the long run.

Question 25

How can the genome of an organism be sequenced?

Answer 25

Only works on sections of 750bp long
Genomes are mapped to identify what part of the genome they have come from
Samples of the genome are sheared into smaller sections of around 100,000bp
These sections are placed into separate bacterial artificial chromosomes (BACs) and transferred to bacterial cells. They clone the sections
DNA is extracted from the cells and restriction enzymes used to cut it into smaller fragments
Fragments are separated by electrophoresis
Fragments are sequenced using an automated process
Computer programs put all the fragment sequences together

Question 26

What are the advantages of gene sequencing?

Answer 26

Knowing the genomes of many different species allows genome wide comparisons between individuals and species. This allows us to do many things, such as identify what genes are essential for life (those found in all organisms); show evolutionary relationships; find what sections of DNA are important in causing disease and identifying mutant alleles/the presence of those that can increase the risk of particular diseases

Question 27

Define recombinant DNA

Answer 27

A section of DNA, often in the form of a plasmid, which is formed by joining DNA sections from 2 different sources/species

Question 28

What does genetic engineering involve?

Answer 28

The extraction of genes from one organism (or the manufacture of genes) in order to place them in another organism, often of a different species, such that the receiving organism expresses the gene product

Question 29

How can sections of a DNA containing a desired gene be extracted from a donor organism?

Answer 29

Restriction enzymes are used to cut DNA at their restriction sites - usually 4-6bp long. Get a ‘sticky end’ - overhanging base pairs. Fragments then separated by electrophoresis and the ones containing the gene are obtained

Question 30

How can DNA fragments be separated by size?

Answer 30

Electrophoresis. The DNA fragments are negatively charged due to the phosphoryl groups. They are placed in a well at one end of an agarose gel, and an electrical current is applied across it through a buffer solution with the wells at the negative end. The DNA fragments move from the negative end to the positive end. The longer fragments get caught up in the agarose gel and therefore move slower and don’t get as far in the time they have, whilst the shorter ones travel much further.

Question 31

How can DNA with specific sequences be identified?

Answer 31

DNA probes: a short, single stranded piece of DNA (50-80bp long) that is complementary to the section of DNA being investigated is marked (either radioactive or fluorescent). Probes anneal to places where the complementary strand is present, and we can find the specific base sequence.

Question 32

How does the polymerase chain reaction work?

Answer 32

Generates multiple copies of a DNA fragment. Works on relatively short DNA sequences and is a cyclical reaction
DNA sample is mixed with DNA nucleotides and DNA polymerase. Heated to 95C to break hydrogen bonds holding complementary strands together. Primers are added (10bp in length). Temp reduced to 55C to allow primers to bind to form small sections of double stranded DNA. DNA polymerase binds to double stranded segments, temp increased to 72*C (optimum temp), and DNA nucleotides are attached to DNA fragments, generating new double stranded molecule. Process is repeated

Question 33

How can isolated DNA fragments be placed in plasmids?

Answer 33

DNA fragments and plasmid cut with the same restriction enzyme so have matching sticky ends, so hydrogen bonds form between complementary strands. Fragments of DNA mixed together with DNA ligase which catalyses the condensation reaction that joins the sugar phosphate backbone.

Question 34

What vectors can DNA be incorporated into?

Answer 34

Plasmids, viruses, yeast cell chromosomes

Question 35

How can plasmids be taken up by bacterial cells?

Answer 35

Plasmids are mixed with the bacterial cells and calcium salts are added as well as heat shock treatment. This weakens the cell surface membrane, allowing plasmids to be taken up. However, less than 1% of the cells will take up a plasmid; these are known as transformed bacteria and results in the bacteria creating new DNA. Therefore, the bacteria are transgenic.

Question 36

Why is it an advantage to microorganisms to be able to take up new plasmids?

Answer 36

This a process known as conjugation, where genetic material is exchanged. Plasmids are swapped between cells that are sometimes of a different species. Speeds the spread of antibiotic resistance between bacterial populations. Conjugation contributes to genetic variation and evolution of survival mechanisms

Question 37

How are genetic markers used to identify transgenic bacteria?

Answer 37

Plasmid is selected as it contains 2 different antibiotic resistance genes. The plasmids are cut in the middle of one of the genes, so that if the required gene is taken up, the bacteria is no longer resistant to antibiotic 2, however the gene for antibiotic 1 still works. The bacteria are grown on an agar plate so the cells form colonies. Antibiotic 1 is used to separate out those that have taken up a plasmid (everyone else dies). A print is taken, and antibiotic 2 is applied to that. The colonies that die have taken up the gene. Go back and get your colonies!! Antibiotic 1 is usually ampicillin and antibiotic 2 is usually tetracycline

Question 38

How is genetic engineering of bacteria used to produce human insulin?

Answer 38

mRNA of the insulin gene is found and the enzyme reverse transcriptase is used to synthesise a complementary DNA strand, which is made into a double strand with DNA polymerase and free nucleotides. We now have a cDNA gene. Unpaired nucleotides are added to give complementary sticky ends to that of the plasmid, which is cut open with a restriction enzyme. cDNA and plasmids mixed with DNA ligase, forming some recombinant plasmids. Plasmids mixed with bacteria and are grown on an agar plate. Those containing the insulin gene are identified and then are grown in continuous culture.

Question 39

Outline the process involved in the genetic engineering of ‘Golden Rice’

Answer 39

Vitamin A deficiency. Vitamin A can be synthesised in body from beta-carotene, found in some plants. Rice is engineered to have beta-carotene gene switched on in part of rice that is eaten - the endosperm. Only need to add 2 enzymes to complete metabolic pathway for production of beta-carotene: phytoene synthetase from daffodils and Crt 1 from Erwinia uredovora. Inserted near a specific promoter sequence that switches the genes on.

Question 40

How can animals be genetically engineered for xenotransplantation?

Answer 40

One of the main problems with xenotransplantation (animal organs into humans) is immune rejection. Animals can be genetically modified to lack some of the genes that trigger the immune response, such as alpha-1,3-transferase. Genes such as human nucleotidase enzyme can also be added.

Question 41

What is gene therapy?

Answer 41

The treatment of genetic disorders with molecular genetic technology. Adding a working copy of a gene into cells may alleviate the symptoms of sufferers. Genes could also be silenced by using mRNA

Question 42

What is the difference between somatic cell gene therapy and germline cell gene therapy?

Answer 42

Somatic cell: 2 types - augmentation and killing specific cells. Augmentation involves adding a copy of the functioning gene to a cell in which only a faulty allele is present. Killing specific cells involves getting cancerous cells to express certain antigens that make them vulnerable to cancer treatment drugs. Functioning allele will not be passed on.
Germline cell: engineering a gene into the sperm, egg or zygote, so the gene is present in all the cells of the organism. The functioning allele may also be passed to the offspring.

Question 43

What are the ethical concerns raised by genetic manipulation?

Answer 43

Engineered microoganisms may escape and transfer genes to others
Antibiotic resistance markers increasing issue of super bugs
Passing crop plant genes to wild relatives resulting in less genetic variation
Passing plant resistance genes to weeds, creating super weeds
Modified plants may be toxic
Resistant plants may cause pathogen attack to get more aggressive
Animal welfare issues; animal suffering
Gene transfer effect unpredictable
Germline gene cell therapy may lead to designer babies