Cloning and biotechnology Flashcards

Question

History of biotechnology

Answer 1

-first coined by Karoly Ereky 1919- used the term to describe any technological process that made use of living organisms or parts of living organisms to manufacture useful products or provide useful services -this included domestication of animals, planting of crops, mechanisation of agricultural processes and selective processes of plants and animals -new science of DNA technology brought biotechnology to its current position- out increasing understanding of genetics and genetic engineering along with ability to manipulate living conditions of living organisms has led huge expansion of biotechnology

Answer 2

-ethanol in beer and wine -carbon dioxide used to make bread rise -lactic acid used to make cheese and yoghurt -mycoprotein- filamentous fungus protein used for vegetarian food -soya- soya beans fermented to produce soy sauce

Answer 3

-penicillin -other antibiotics -insulin, other therapeutic human proteins

Answer 4

-protease and lipase used in washing powders -pectinase used to extract juice from fruit -sucrase to digest sugar to make food sweeter -amylase to digest starch into sugar to produce syrup used as sweetener -protease used to tenderise meat -lactase to make lactose free milk -removing sticky residues from recycled paper

Answer 5

-biogas, combination of carbon dioxide and methane -citric acid, food preservative -bioremediation, cleaning waste water

Answer 6

-relatively cheap and easy to grow microorganisms -production processes can take place at lower temperatures saving fuel and reduce costs -production process can take place at normal atmospheric pressures, safer than chemical reactions which may require very high pressure -production process not dependant on climate- can take place anywhere with resources to build and run suitable equipment -microorganisms can be fed by products of other food industries, e.g. starch, waste or molasses, but need to be pre treated -microorganisms have short life cycle and reproduce quickly- large population can grow very quickly inside fermenter -microorganisms can be genetically modified easily which allows very specific production processes to be achieved -fewer ethical considerations to worry about in using microorganisms -products often released from microorganism into surrounding medium- makes product easy to harvest

Answer 7

-genetically modified mammals e.g. sheep, goats and cows can be used to produce useful proteins -in some mammals, proteins are incorporated into milk and easily harvested -e.g. goats have been genetically modified to possess gene for spider silk and secrete it into they milk -other cases, protein may be secreted into blood -e.g. GM cows synthesise human antibodies which can be isolated from their blood

Answer 8

-gene technology -genetic modification and gene therapy -selective breeding -cloning by embryo splitting and micropropagation -use of enzymes in industrial processes -immunology

Answer 9

-yoghurt is milk that has undergone fermentation by Lactobacillus bulgaricus and Streptococcus thermophilus -bacteria convert lactose to lactic acid -acidity denatures the milk protein causing it to coagulate -bacteria partially digest the milk, making it easy to digest -fermentation also produces flavour characteristic of yoghurt -other bacteria may be added as probiotics- bacteria which may benefit human health by improving digestion of lactose, aiding gastrointestinal function and stimulating the immune system

Answer 10

-milk is usually pre treated with culture of bacteria that can produce lactic acid from lactose -once acidified, milk is mixed with rennet -rennet contains enzyme rennin (chymosin) found in stomachs of young mammals, however now we use GM bacteria -rennin coagulates milk protein (casein) in presence of calcium ions 1) Kappa casein which keeps casein in solution is broken down- this makes casein insoluble 2) casein is precipitated by action of calcium ions which bind molecules together -resulting solid called curd is separated from liquid component (whey) by cutting. stirring and heating -bacteria continue to grow producing more lactic acid -curd is then pressed into moulds

Answer 11

-treatment whilst making and pressing curd determines the characteristic of the cheese -flavour is determined during later ripening and maturation processes -cheese can be given additional flavour by inoculation with fungi such as Penicilum to produce blue cheese

Answer 12

-bread is mixture of flour, water and salt with some yeast (single celled fungus S. cerevisae) 1) mixing- ingredients are mixed together thoroughly by kneading and this produces a dough 2) proving/ fermenting- dough is left in warm place for up to 3 hours whilst yeast respires anaerobically- this produces carbon dioxide bubbles causing dough to rise 3) cooking- risen dough is baked. Any alcohol evaporates during the cooking process -two proteins- gliadin and glutenin form gluten when mixed together -gluten gives dough elastic properties and allows CO2 to be trapped

Answer 13

-alcoholic beverages are product of anaerobic respiration of yeast -wine is made using grapes that naturally have yeasts on their skin -grapes contain the sugars fructose and glucose -when the grapes are crushed the yeast uses these sugars to produce carbon dioxide and alcohol -ale or beer is brewed using barley grains beginning to germinate -as grain germinates it converts stored starch to maltose which is respired by the yeast -anaerobic respiration again produces carbon dioxide and alcohol -hopes are used to give bitter taste to liquid

Answer 14

-more recently microorganisms used to manufacture protein directly used as food -mycoprotein marketed as meat substitute for vegetarians and a healthy option for non vegetarians as it contains no animal fat or cholesterol -these fungi can produce protein with similar amino acid profile to animal and plant protein -they can grow on almost any organic substrate including waste materials such as paper and whey -must add nitrogen so that amino acids and nucleic acids can form the protein

Answer 15

-production protein faster than that of animal or plant protein -biomass produced has very high protein content 45-85% -production can be increased and decreased according to demand -no animal welfare issues -microorganisms easily genetic modified to adjust amino acid content -SCP production could be combined with removal of waste -production is independent of seasonal variations -not much land is required

Answer 16

-may not want to eat fungal protein or food grown on waste -isolation of protein- microorganisms need to be isolated in huge fermenters from material they grow on -protein need to be purified to ensure uncontaminated -microbial biomass may have high proportion nucleic acids which must be removed -amino acid profile may be different from traditional animal protein- e.g. deficient in methionine -infection- conditions needed for microorganisms to grow are also ideal for pathogenic organisms -palatability- protein does not have taste or texture of traditional protein sources

Answer 17

-commercial drug production uses large stainless steel containers called fermenters in which growing conditions can be controlled to ensure best possible yield of product -temperature- too hot= enzymes denature, too cold= growth limited -nutrients available= microorganisms require nutrients to grow and synthesise the product. Sources of carbon, nitrogen, minerals and vitamins are needed -oxygen availability- most microorganisms respire aerobically -pH- enzyme activity and hence growth and synthesis are affected by extremes of pH -concentration of product- if product builds up, may affect synthesis -a fermenter must first be sterilised using superheated steam -to can then be filled with all components for growth and supplied with a starter culture of microorganism to be used -culture will be left to grow and synthesise products

Answer 18

-pressure vent prevents any gas build up -air inlet- sterile air provide oxygen in aerobic fermenters -mixing blades (impellers) -motor- rotates blades to mix culture evenly -water jacket inlet- allows circulation of water around fermenter to regulate temperature -outlet tap for draining fermenter -inlet for addition of nutrients -electronic probes to measure oxygen, pH and temperature levels -air outlets often in ring- air bubbles from outlets mix with culture (known as sparging) -all inlets and outlets contain filters to prevent contamination

Answer 19

-some products synthesised by microorganism during normal metabolism when actively growing- called primary metabolites -such products are continually released from cells and can be extracted continuously from fermenting broth -broth is topped with nutrients as are used by microorganisms otherwise population becomes too dense -this is known as continuous culture and keeps microorganism growing at specific growth rate

Answer 20

-other products are produced only when cells are placed under stress such as high population density or limited nutrient availability- these are called secondary metabolites and are produced mostly during stationary phase of growth -hence culture set up with limited quantity of nutrients and allowed to ferment for a specific time -after this time fermenter is emptied and product can be extracted from culture- this is known as batch culture

Answer 21

-asepsis is ensuring sterile conditions are maintained -nutrient medium would also support growth of unwanted microorganisms which would reduce production because unwanted microorganisms: -complete with cultured microorganisms for nutrients and space -reduce yield of useful products -spoil the product -produce toxic chemicals -destroy cultured microorganisms and their products -in processes where food or medicinal chemicals are produced, all products must be discarded if contamination by unwanted organisms occurs

Answer 22

-Florey and chain devised process to successfully mass produce penicillin through fermentation by fungus Penicillum chrysogenum -modern strains of fungus have been selectively bred to be more productive than early strains -penicillin is secondary metabolite- only produced once population has reached certain size -therefore penicillin manufactured by batch culture 1) fermenter runs for 6-8 days. Culture then filtered to remove cells 2) Antibiotic precipitated as crystals by addition of potassium compounds. Antibiotic may be modified by action of other microorganisms or by chemical means 3) Antibiotics mixed with inert substances and prepared for administration in tablet form or as syrup or in form suitable for injection

Answer 23

-insulin widely used to treat type 1 diabetes -previously extracted from pancreas of animals such as cattle or pigs sent for slaughter -insulin from slaughtered animals is not identical to human insulin and so less effective than human insulin and expensive to extract -1978 synthetic human insulin developed by genetically modifying bacterium -gene for human insulin was combined with plasmid to act as vector so gene could be inserted into bacterium E.coli -the resulting genetically modified bacterium enabled production of vast quantities of human insulin at relatively low cost -insulin manufactured by continuous culture

Answer 24

-bioremediation is use of microorganisms to clean soil and underground water on polluted sites -organisms convert toxic pollutants to less harmful substances -involves stimulating growth of suitable microbes that use contaminants as source of food -requires right conditions for growth of microorganisms: available water, suitable temperature and pH -where conditions not quite suitable may be modified through addition of suitable substances -additional nutrients such as molasses may be needed to ensure microorganisms can grow effectively -may be necessary to pump oxygen in for aerobic bacteria -where conditions cannot be made suitable in situ, soil may be dug up and moved to be treated ex situ

Answer 25

-uses natural systems -less labour/ equipment required -treatment in situ -few waste products -less risk of exposure to clean up personnel -HOWEVER bioremediation only suitable for certain products; heavy metals such as cadmium and lead cannot be treated

Answer 26

-microorganisms will grow on any material that provides carbon compounds for respiration and source nitrogen for protein synthesis -however in lab, microorganisms are grown in growth medium: -soup like liquid broth, kept in bottles or tubes OR -set, jelly like substances called agar, melted and poured into Petri dishes -typical nutrient agar contains peptones (from enzymatic breakdown of gelatine), yeast extract, salts and water -may also contain glucose or blood

Answer 27

-aseptic techniques have been developed to reduce likelihood of contaminating medium with unwanted bacteria or fungi 1) wash your hands 2) disinfect working area 3) have bunsen burner operating nearby to heat air. This causes air to rise and prevent air borne microorganisms settling. Also creates area around it of sterile air in which microbiologist can work 4) As open vessel, pass neck of bottle over flame to prevent bacteria in air entering bottle. Bottle should also be flamed as it is closed 5) do not completely lift lid off Petri dish- just open it enough to allow introduction of desired microorganism 6) any glassware or metal equipment should also be passed through flame before and after contact with desired microorganisms

Answer 28

-microorganisms can grow almost anywhere -key is to ensure they grow on medium used and grow desired microorganism rather than others that have infected medium by mistake -growing microorganisms on agar plates involved 3 main steps 1) sterilisation 2) inoculation 3) incubation

Answer 29

-nutrient agar medium and any equipment used must be sterilised -medium sterilised by heating in an autoclave at 121C for 15 minutes (high temperature achieved by boiling water under high pressure inside autoclave) -this kills all living organisms including any bacterial or fungal spores -when medium cooled sufficiently to handle it is poured into sterile Petri dishes and let to set -it is important lid is kept on Petri dish to prevent infection

Answer 30

-inoculation is the introduction of microorganism to the sterile medium- this can be achieved many ways: -streaking- a wire inoculating loop is used to transfer a drop of liquid medium onto surface of agar. The drop is drawn out into streak by dragging the loop across surface. Take care not to break the surface of agar -seeding- a sterile pipette can be used to transfer a small drop of liquid medium to the surface of the agar to the Petri dish before the agar is poured in -spreading- sterile glass spreader may be used to spread the inoculated drop over the surface of agar -a small cotton swab or cotton bud can be moistened with distilled water and used to collect microorganisms from a surface and then carefully wiped over the surface of agar medium

Answer 31

-Petri dish must be labelled and top must be taped to bottom using 2 strips of adhesive tape- be careful not to seal Petri dish completely -as this can lead to selection of anaerobic bacteria which may be pathogenic -Petri dish then placed in suitable warm environment such as incubator -should be placed upside down as this prevents drops of condensation falling onto surface of agar -also prevents agar medium from drying out too quickly -suitable temperatures will depend on type of microorganisms being grown -cultures can be examined after 24-36 hours -do not open Petri dish -all petri dishes must be completely sterilised after use and before disposal -thoroughly wash hands after handling petri dish as any moisture coming out of dish could be source of infection

Answer 32

-bacteria grow in visible colonies which may be shiny or dull -some colonies are round with entire edges, while others have crenate edges -colonies can also be range of different colours -each colony results from a single bacterium -filamentous fungi grow into mass of hyphae which may also be circular but mass is not shiny and often looks like cotton wool with fluffy aerial hyphae -single celled fungi (yeasts) grow as circular colonies

Answer 33

-liquid broth initially clear but will turn cloudy when bacteria have grown -a liquid broth can be useful to increase the numbers of microorganisms before transferring to agar plates for counting or identification -similar aseptic techniques as described before must be applied when using liquid broth -liquid broth can be used to identify population growth

Answer 34

-culture which has no exchange of nutrients or gas with external environment

Answer 35

-sequence of dilutions used to reduce concentrations of a solution or suspension

Answer 36

-a liquid broth can be used to measure growth rate of microorganism population -sterile broth inoculated and population size measured at regular intervals of incubation -population size can be measured by transferring a small sample to an agar plate and incubating agar culture- each individual microorganisms will produce a visible colony

Answer 37

-its stepwise dilution of broth culture -at each step, broth diluted by factor of 10 -take 1cm3 sample of broth and add 9cm3 of distilled water- label as 10-1 -take a 1cm3 sample of this diluted broth and add 9cm3 of distilled water- label as 10-2 -continue procedure until series of dilutions with suitable labels -a drop of dilution can be used to inoculate agar plate -one of them will produce culture plate in which number of colonies can be counted -when recording population density, do not forget to multiply count by dilution factor and also by added volume to plate

Answer 38

-numbers of individual microorganisms in a broth can be high -if broth used to inoculate agar plate, may be too many colonies which merge together making counting impossible -in order to investigate rate of growth of population of microorganisms its essential to reduce population density- achieved by serial dilution

Answer 39

-small population of microorganisms in closed culture that contains all the nutrients required for growth will undergo population growth -a closed culture refers to population in which conditions set at start and no exchange with external environment -population growth will follow a predictable pattern -these are similar to conditions set up for batch production in a fermenter -in batch production however certain substances such as oxygen may be added to keep population growing until nutrients used up

Answer 40

LAG PHASE -in early population growth population does not grow quickly because population still small and organisms adjusting to environment -involves taking up water, cell growth, activating genes, synthesising enzymes LOG (EXPONENTIAL) PHASE -in log phase organisms have adjusted to environment -they each have enzymes needed to survive -each individual has sufficient nutrients and space to grow rapidly and reproduce -population doubles in size with each generation STATIONARY PHASE -eventually increasing numbers of organisms use up nutrients and produce increasing amount of waste products such as carbon dioxide and other metabolites -rate of population growth declines and number of individuals dying increases until reproduction rate equals death rate -this is stationary phase where there is no population growth DEATH (DECLINE) PHASE -nutrients run out and concentration of waste products may become lethal -more individuals die than are produces and population begins to fall -eventually all organisms will die

Answer 41

-primary metabolites produced during normal activities of microorganism will be collected from fermenter during log phase -in fermenter population not kept in closed culture but conditions are maintained for optimal growth

Answer 42

-secondary metabolites are produced in stationary phase -population must be kept in closed culture and metabolites can be collected at end of stationary phase or during decline phase

Answer 43

-some biotechnology processes can be simplified by taking enzymes out of microorganisms -enzymes are large proteins that act on substrates to generate the product -enzymes are not used up in the reaction and remain in suspension when reaction has been completed -in industrial process this means product must be isolated from enzymes before use -this could be expensive

Answer 44

ADVANTAGES -do not mix with the product therefore extraction costs are lower -continuous processes made easier as there are no cells requiring nutrients, reproducing and releasing waste products- no need for down streaming -enzymes are surrounded by immobilising matrix which protects them from extreme conditions- so higher temperatures or wider pH range can be used without causing denaturing -can be recycled/ reused DISADVANTAGES -initial time and materials required to immobilise the enzyme may raise costs -fewer collisions as active site may be hidden therefore lower rate of reaction -leakage may be high

Answer 45

-enzyme molecules are bound to a supporting surface by a combination of hydrophobic and ionic links -however these bonds are weak and therefore there may be high leakage -suitable surfaces include clay, porous carbon, glass beads and resin -enzyme molecules are bound with active site exposed and accessible to substrate- higher rate of reaction

Answer 46

-enzyme molecules are bonded to a supporting surface such as clay using strong covalent bonds -enzymes are bonded using cross linking agent, which may also link them in a chain -the production of covalent bonding can be expensive and distort enzyme active site, reducing activity -however enzymes are much less likely to become detached and leak into reaction mixture

Answer 47

-enzyme molecules are trapped in matrix that does not allow free movement -enzyme molecules are unaffected by entrapment and remain fully active -however, substrate molecules must diffuse into entrapment matrix and product molecules must be able to diffuse out -method therefore suitable only for processes where substrate and product molecules are relatively small - slows the rate of reaction -may use gel beads or a network of cellulose

Answer 48

-enzyme molecules separated by reaction mixture by partially permeable membrane -as in entrapment substrate and product molecules must be small enough to pass through partially permeable membrane by diffusion -this access to enzymes may limit reaction rate

Answer 49

-converts glucose to fructose -probably most widely used enzyme because number of applications of syrup used -used to produce high fructose corn syrup (HFCS) which is much sweeter than sucrose -HFCS often used in diet foods as less sugar needs to be added for equivalent sweetness -may also be used as sweetener in foods for diabetics -HFCS much cheaper than sucrose and so is widely used in food industry to replace sucrose e.g. in soft drinks, jam, icecream, cereals etc

Answer 50

-formation semi synthetic penicillins e.g. amoxicillin, ampicillin which were first developed during 1960s -some microorganisms not resistant to these forms of semi synthetic penicillins

Answer 51

-convert lactose to glucose and galactose by hydrolysis- used to produce lactose free milk -milk important source of calcium needed for strong bones and teeth -people with insufficient calcium in diet more likely to develop weak bones or osteoporosis -therefore important people that are lactose intolerant given calcium supplements or lactose free milk

Answer 52

-converts dextrins to glucose -during hydrolysis of starch short polymers of glucose (dextrins) are formed -hydrolysis by glucoamylase can convert these dextrins to glucose -glucoamylase can be immobilised on variety of surfaces and used to digest sources of starch such as corn and cassava -the enzyme used in wide range of fermentation processes including conversion of starch pulp to alcohol used to produce gasohol- an alternative fuel for motor vehicles -also used within food industry to make high fructose corn syrup

Answer 53

-cut mini floret from cauliflower -cut the floret into small 3mm pieces- these are the explants -transfer explants into sterilising solution and swirl solution periodically- take care of inhalation or skin contact as irritant -after 15 minutes transfer explants onto sterile agar surface in specimen tube -important to use aseptic techniques to avoid contaminating the explants during this stage -place cap on tube -incubate in warm lab -growth should be visible within two weeks

Answer 54

-use aseptic techniques throughout and avoid contact with bacteria -use commercially supplied colony of bacteria to inoculate nutrient broth in bottle -thoroughly shake broth and incubate at 25C -at zero time take small sample of broth using sterile pipette that gives standard size drop -inoculate agar plate and use sterile glass spreader to spread drop over agar surface -at intervals 2 hours shake broth and take another sample to inoculate another nutrient agar plate -when broth looks cloudy, carry out serial dilution and use each dilution to inoculate a sterile agar plate -incubate plates at 25C -24hours record number of bacterial colonies visible on each plate and multiply by any dilution factor used -use volume of drop added to plate to calculate density of cells -plot population density against time, typically y axis should be log10 scale

Answer 55

BINARY FISSION -cell elongates and DNA replicated -new DNA goes through conjugation tube -cell wall and plasma membrane begin to divide (cytokinesis) -cross walls form completely around DNA -genetically identical cells

Answer 56

MITOSIS INTERPHASE -G1- organelles replicated -S - DNA replicated -G2- energy stores increase -enzyme checkpoints between each PROPHASE -DNA supercoils, condenses- chromosomes visible -nuclear envelope breaks down -centrioles to poles METAPHASE -chromosomes line up along equator ANAPHASE -chromosomes pulled apart -chromatids go to poles TELOPHASE -nuclear envelope reforms -spindle fibres break down CYTOKINESIS

Answer 57

-they have lots of meristematic tissue- e.g. apical buds, roots and cambium- which is pluripotent -humans have multipotent stem cells which are too differentiated to form whole new organism

Answer 58

PARTHENOGENESIS -e.g. in sharks, snails, komodo dragons -its eggs can become fertilised and produce clones of itself -advantageous as when population numbers low its hard to find mates- do not need to find mates -males aggressive- females less likely to get injured -quick way to reproduce- no meiosis, do not need to find mate FRAGMENTATION -e.g. in starfish, if they lose a limb they can reform the lost limb

Answer 59

-aerobes= organism that requires oxygen to survive -obligate anaerobes= won't survive in oxygen environment

Answer 60

-temperature rise and barley seed starts to absorb water -this stimulates the embryo to produce gibberellins -gibberellin molecules diffuse into the aleurone layer and stimulate the cells there to synthesise amylase -the amylase hydrolyses starch molecules in the endosperm, producing soluble maltose molecules -the maltose is converted to glucose and transported to the embryo -glucose can be respired by the embryo, providing the embryo with the energy needed for it to grow

Answer 61

ADVANTAGES -more frequent collisions with substrate so high rate of reaction -can be collected via extracellular enzymes DISADVANTAGES -protease enzymes can breakdown the enzymes -have to be removed downstream (mixed in with substrate so needs to be extracted/ purified)- increases the cost

Answer 62

ADVANTAGES -good for use when there is a series of enzymes in the reaction -no extraction- less expensive -used when enzyme is unstable/ inactive when removed from cell DISADVANTAGES -some of substrate is used by cell- e.g. for respiration -risk of contamination - you can use the biuret test to test for proteins to see if there has been contamination -expensive to maintain the microorganism

Answer 63

-isolated= able to freely mix with substrate -immobilised= bound to a support -whole cell= whole cells that contain enzymes, may be immobilised or used free

Answer 64

final population= initial population x 2n log2(final population/ initial population)= n n= number of cycles/divisions