Biotechnology & Gene Technologies Module 2 Biotechnology & Gene Technologies

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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)

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

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

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