1. Biotechnology & Gene Technologies
Module 2
Biotechnology & Gene Technologies

2. Difference between reproductive and non-reproductive cloning
Reproductive: produce new organisms
Non-reproductive: generate cell, tissues or organs

3. Production of Natural Clones
For example: vegetative propagation in elm trees
Reproduces asexually following damage to the parent plant
Basal sprouts
Grow from meristem tissue in the trunk close to the ground

4. Production of artificial clones: tissue culture
Small piece of tissue taken from plant to be cloned (explant)
Placed in growth medium
Cells divide by don’t differentiate forming a callus
Cell is removed from callus and placed on growth medium containing hormones
Plantlets can then by propogated

5. Plant cloning in agriculture
Advantages
Grow plants from sterile plants
Know features produced: yield, taste, colour, disease-resistance
Cost is reduced
Faster than selective breeding
Large numbers can be generated from single valuable plants
Disadvantages
Genetic uniformity means all plants are equally susceptible to pest, disease or environmental change

6. How artificial clones of animals can be produced
Cloning by splitting embryos
Egg and sperm used in in vitro fertilisation
Grow to 16 cell embryo
Split into several embryos
Implant into surrogates
Cloning by nuclear transfer (Dolly)
Take an ovum and remove the nucleus to create enucleate ovum
Take adult body cell
Use electro-fusion to fuse enucleate ovum and adult body cell
‘Culture’ in tied embryo of sheep
Recover early embryo and implant into a surrogate

7. Cloning animals Advantages Disadvantages
High value animals can be cloned in large numbers
Rare animals can be cloned to preserve the species
Genetically modified animals can produce pharmaceutical chemicals in the milk
Disadvantages
High value animals not necessary good for animal welfare
Genetic uniformity means they can’t adapt to changes in the environment
Unknown if animals cloned from adults remain healthy in the long term

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

9. Microorganisms are often used in biotechnological processes
Grow rapidly in favourable conditions
Produce proteins into surrounding medium
Can be genetically engineered to make specific products
Grow well a relatively low temperatures
Can be grown anywhere in the world
Generate pure products

10. Standard growth curve of a micro organism in a closed culture
Lag Phase
Organisms are adjusting to the surrounding conditions
Log Phase
Population size doubles every generation
Stationary Phase
Nutrient levels decrease and waste products increase
Rate of death matches rate at which new individuals form
Death Phase
Nutrient levels fall so low and waste products increase so much death rate is higher than formation of new individuals

11. Enzymes can be immobilised
Adsorption
Enzyme molecules mixed with immobilising support
Binds to support by hydrophobic interactions and ionic links
E.g. clay, glass beads, resins
Disadvantage: enzymes can become detached
Advantages: enzymes active site is displayed
Covalent bonding
Enzymes are covalently bonded to a support
Enzymes covalently bonded to an insoluble material by cross linking agent e.g. gluteraldehyde
Advantage: very little leaking of enzymes from support
Disadvantage: doesn’t immobilise large number of enzymes
Entrapment
Enzymes trapped in gel or cellulose fibres
Advantage: Active site not affected by entrapment
Disadvantage: substrate molecules have to get through barrier
Membrane separation
Enzyme solution held on one side of partially permeable membrane and substrate is passed along other side
Advantage: Products small enough to pass back through membrance
Disadvantage: any contamination is costly to deal with

12. Immobilised Enzymes Why used in large scale production
Enzymes not present with products so purification costs are low
Enzymes are immediately available for reuse
Immobilised enzymes are more stable
Enzymes can catalyse specific reactions
Enzymes function well at relatively low temperatures

13. Processes of continuous & batch culture
Continuous culture
Industrial scale fermentation
Nutrients added to fermentation tank and products removed at regular intervals
Batch culture
Starter culture of microorganisms mixed with specific quantity of nutrients
At end of the period products are removed and fermentation tank is emptied

14. Difference between primary and secondary metabolites
Substances produced by an organism as part of growth e.g. amino acids
Production of primary metabolites matches growth of population
Secondary
Substances produced by an organism that are not part of its normal growth e.g. antibiotic chemicals
Production of secondary metabolites begins after main growth period so growth doesn’t match population

15. Maximum yield of product in a fermentation vessel
Manipulating growing conditions
Temperature
Too hot and enzymes denature
Too cold and growth is slow
Type and time of addition of nutrient
Addition of nutrients can be timed to produce primary or secondary metabolites
Oxygen concentration
Need growth of organisms in aerobic conditions
Don’t want unwanted anaerobic products pH
Can reduce activity of enzymes and therefore growth rates

16. Importance of asepsis in the manipulation of microorganisms
Don’t want unwanted microorganisms (contaminant)
Compete for nutrients and space
Reduce yield
May cause spoilage of products
May produce toxic chemicals
May destroy culture micro organisms and their products

17. Sequencing the genome of an organism
Genome must be broken down and sequenced in sections, sequence must be carried out several times in overlapping fragments
Genomes mapped to identify which part of genome they come from
Genome is cut into sections
Sections placed into separate bacterial artificial chromosomes (BACs), transferred into E. coli and replicated up (clone libraries)
DNA extracted from BACs
Restriction enzymes cut into fragments
Fragments separated by gel electrophoresis
Each fragment sequenced
Computer programmes compare overlapping fragments

18. How gene sequencing allows for genome-wide comparisons between individuals and between species
Allows comparison of genes for same proteins across range of organisms
Shows evolutionary relationships
Can test effect of mutations
Help develop more effective vaccines/drugs
Analyse individuals for particular diseases

19. Define: recombinant DNA
A section of DNA, often in the form of a plasmid, which is formed by joining DNA sections from two different sources

20. Genetic Engineering
Involves the extraction of genes from one organism, or the manufacture of genes, in order to place in another organism (often a different species) such that the receiving organism expresses the gene product

21. DNA containing a desired gene can be extracted from a donor organism
mRNA can be extracted from cell and used as a template to make a copy of the gene
DNA probe used to locate gene and DNA fragment cut out using restriction enzymes
Cut DNA at specific points
Cuts at specific restriction site
Gives a staggered cut known as a sticky end

22. Gel Electrophoresis DNA fragments can be separated by size
DNA cut into fragments by restriction enzymes
DNA samples placed into wells
Electric current placed through gel
DNA is attracted to positive electrode
Shorter DNA moves faster than longer
Position of fragments shown by DNA dye

23. DNA Probes
DNA probes can be used to identify fragments containing specific sequences
DNA probes are short single stranded piece of DNA complementary to a section of DNA being investigated, they anneal to the complementary bases so they can be located
DNA probe is labelled
Radioactive marker
Fluorescent marker

24. Polymerase Chain Reaction (PCR)
Can be used to make multiple copies
DNA, DNA nucleotides and DNA polymerase mixed together
Heated to 95 degrees to separate double stranded DNA into single strands
Primers added (short single DNA strands)
Temperature reduced to 55 degrees so primers bind to the single stranded DNA
DNA polymerase binds to the double stranded sections
Temperature raised to 72 degrees, DNA polymerase extends double stranded sections
Repeat

25. Plasmids Isolated DNA fragments can be placed in plasmids
DNA ligase catalyses condensation reaction joining sugar phosphate back bone of DNA of complementary sticky ends

26. Vectors Into which DNA fragments may be incorporated Plasmid
Virus Genomes
Yeast cell chromosomes

27. Plasmids
May be taken up by bacterial cells in order to produce a transgenic microorganism that can express a desired gene produce
Large quantities of plasmids mixed with bacterial cells
Add calcium salts
Heat shock: drop temperature to freezing and quickly raise to 40 degrees

28. Microorganisms with the capacity to take up plasmid DNA from the environment
Advantages
Plasmids often contain antibiotic resistance gene
Increased genetic variation

29. Genetic Markers
Genetic markers in plasmids can be used to identify the bacteria that have taken up a recombinant plasmid
Original plasmid contains genes for resistance to two different antibiotics
Restriction site in middle of tetracycline gene so addition of gene removes resistance to tetracycline
Replica plating is used
Bacteria grown on nutrient gel
Cells from each colony transferred onto ampicillin gel, only those which have taken up plasmid will grow
Cells from those colonies transferred onto tetracycline gel, only those whose plasmids haven’t got the gene will die

30. Genetic engineering of bacteria to produce human insulin
mRNA isolated and treated with reverse transcriptase to make cDNA (copies of human insulin gene)
Add sticky ends complementary to plasmid
DNA ligase seals gene into plasmid (plasmid taken from bacteria and cut with restriction enzyme)
Bacteria take up recombinant plasmid

31. Genetic engineering of golden rice
Makes beta carotene accumulate in rice
Phytoene synthase enzyme from daffodil is added to rice (makes phytoene)
Crt1 enzyme gene from bacterium added to rice (makes lycopene from phytoene)
Enzymes already present in the rice turn lycopene into beta carotene

32. Genetic engineering of animals for xenotransplantation
Genetically engineered to
lack enzyme alpha 1,3 transferase (which triggers human rejection)
Have human nucleotidase enzyme (to reduce activities involved in rejection)

33. Define: Gene Therapy
Molecular genetic technology used to treat genetic disorders

34. Difference between somatic cell gene therapy and germ line cell gene therapy
Add functioning copy of gene
Kill specific cells
Makes cancer cells express antigen so immune system attacks them
Short lived treatment
Germline cell gene therapy
Genetically engineer sperm/egg/zygote
So affects all cells in the body
Could be passed onto offspring

35. Ethical Concerns: raised by genetic manipulation of animals (including humans), plants and microorganisms
Use of animals is unethical
Lack of long term knowledge so unknown side effects
Thought as by some as ‘unnatural’, a step too far from selective breeding
Individuals resulting from germline gene therapy have had no say in the altering of their DNA
Slippery slope: germline used to enhance favourable characteristics