6.4- Cloning and biotechnology Flashcards
1
Q
Define clones
A
Genetically identical copies of organisms or cells
2
Q
Describe natural clones
A
- produced by asexual reproduction
- nucleus divides by mitosis- creates 2 identical copies of the DNA, which are then separated into 2 genetically identical nuclei before the cell divides to form 2 genetically identical cells
- the cells may not be physically or chemically identical as after division, they may differentiate to form 2 different types of cells
- offspring are genetically identical to the parent
- e.g. single celled yeasts reproduce by budding, bacteria reproduce by binary fission- both involve exact replication of DNA so cells produced are genetically identical
3
Q
What are advantages of reproduction by cloning (natural cloning)
A
If the conditions for growth are good for the parent, - if growth conditions are good for parent, they will be good for offspring as well
- relatively raid- population can increase quickly yo take advantage pf suitable environmental conditions
- reproduction can occur even if only 1 parent and sexual reproduction not possible
4
Q
What are disadvantages of reproduction by cloning (natural cloning)
A
- offspring may become overcrowded
- no genetic diversity (except that caused by mutation during DNA replication)
- population shows little variation
- selection not possible
- if environment changes to be less advantageous, whole population is susceptible
5
Q
How does natural cloning in plants occur
A
vegetative propagation
6
Q
Outline vegetative propagation
A
- differentiation of plant cells not as complete as that in animals
- means many parts of plant contain cells that retain ability to divide and differentiate into a range of types of cell
- means plants are able to reproduce by cloning
- VP is the process of reproduction through vegetative parts of the plant, rather than through reproductive structures
7
Q
Name examples of natural cloning in plants
A
- runners/stolons/rhizomes/suckers
- bulbs
- corms
- leaves
- tubers
8
Q
Describe runners/stolons/rhizomes/suckers
A
- many plants grow horizontal stems that can form roots at certain points
- called runners or stolons if grow on surface of ground, and rhizomes if underground
- some rhizomes adapted as thickened over-wintering organs from which 1 or more new stems will grow in the spring
- suckers- new stems that grow from he roots of a plant- may be close to the base of an older stem or could be distance away- original horizontal branch may die, leaving the new stem as a separate individual
9
Q
Describe bulbs
A
- e.g. nions
- over-winetring methods for many perennial monocotyledonous plants
- consist of an underground stem from which grow series of fleshy leaf bases
- apical bud- grows into new plant in spring
- often bulb contains more than one apical bird and each will grow into new plant
10
Q
Describe Corms
A
- underground stem with scaly leaves an buds
- remain in ground over winter
- in spring, buds grow to produce one or more new plants
- solid, rather than fleshy like bulb
- croci and gladioli reproduce using corns
11
Q
Describe leaves (natural cloning)
A
- Kalanchoe plant reproduces asexually, as clones grow on lead margins
- immature pants drop off leaf and take root
12
Q
Describe tubers
A
- type of underground stem
- e.g. potatoes- one potato will grow into one or more plants
- each new plant can then produce many new tubers later that year
13
Q
Describe cloning in animals
A
- mammals- clone when identical twins are formed- occurs when fertilised egg (zygote) divides as normal, but the 2 daughter cells split to become 2 separate cells- each cell grows and develops into new individual
- water flea and greenfly- animals that commonly reproduce asexually to produce clones
14
Q
name cloning techniques in plants
A
- cuttings
- tissue culture/micropropagation
15
Q
What is the simplest way to create plant clones
A
Cuttings
16
Q
Describe the process of cuttings
A
1) cut stem between 2 nodes (leaf joints)
2) place the cut end of the stem in moist soil
- new roots will grow from the tissues in the stem- usually from the node, but may grow from other parts of the buried stem
- can be used to make large numbers plants very quickly
17
Q
What can you do if plants don’t take root easily in cutting
A
- dip the cut stem in rooting hormone- helps to stimulate growth
- may also be helpful to wound/remove the bark from the cut end of the stem- encourages plant to produce callus
18
Q
Where else in a plant can cuttings be successfully made from
A
- root cuttings- section of root buried just below soil surface- produces new shoots
- scion cutting- dormant woody twigs
- leaf cuttings- leaf placed on moist soil- develops new stems and new roots- some leaves ,ay produce many new plans from one cutting
19
Q
What is the issue with large-scale cloning if done through taking cuttings, what can be used to get around this
A
- time consuming, needs lots of space, some plants don’t respond well to taking cuttings
- tissue culture techniques are used instead- e.g. how many commercially grown houseplants are cloned
20
Q
Describe tissue culture
A
- series of techniques used to grow cells, tissues, or organs from a sample of cells/tissue
- carried out in nutrient medium under sterile conditions
- application of plant growth substances at correct time can encourage the cells in the growing tissue to differentiate
- widely used commercially to increase the number of new plants in micropropagation
21
Q
What is micropropagation
A
- involves taking a small piece of a plant tissue (the explant) and using plant growth substances to encourage it ti grow and develop into a whole new plant
22
Q
Steps of micropropagation
A
1) suitable plant ,material selected and cut into small pieces- explants
2) explants sterilised using bleach or alcohol- essential to kill any bacteria and fungi as would thrive in conditions supplied to help the plant grow
3) explants placed on sterile growth medium (usually agar gel) containing suitable nutrients- stimulates cells of each explant to divide by mitosis to form a callus
4) once callus has formed, it is divided to produce a larger number of small clumps of undifferentiated cells
5) these small clumps of cells are stimulated to grow, divide and differentiate into different plant tissues- achieved by moving the cells to different growth media- causes roots then shoots to form
6) once tiny platelets have been formed, these are transferred to a greenhouse to be grown in compost or soil and acclimatised yo normal growing conditions
23
Q
Describe explants (micropropagation)
A
- could be tiny pieces of leaf, stem, root, or buyd
- meristem tissue often used as this is always free from virus infection
24
Q
Describe the agar gel (or other growth medium) used in early stages of micropropagation
A
- contains suitable nutrients e.g. glucose, amino acids and phosphate
- also contains high concentrations of auxin and cytokinin- plant growth substance
25
What is a callus (plant cloning)
A mass of undifferentiated, totipotent cells
26
Describe the growth media used after callus has been divided (micropropagation)
- each medium contains different ratios of auxin and cytokinin
- first medium contains 100 auxin : 1 cytokinin- stimulates roots to from
- second medium contains 4 auxin :1 cytokinin- stimulates shoots to form
27
Describe an example of usage of tissue culture techniques
- seed banks- use TC techniques to store plants ay a growth stage when they are not too large- important in conservation of species whose seeds don't remain viable for long periods
28
Advantages of artificial cloning in plants
- relatively rapid methods of producing new plants compared with growing plants from seed
- can be carried out where sexual reproduction isn't possible- plants that have lost ability to breed sexually can be reproduced e.g. commercially grown bananas
- plants that are hard to grow from seed can be reproduced e.g. orchids for the horticulture industry
- plants selected will all be genetically identical to parent plant will display same desirable characteristics e.g. high yield, resistance to common pest/disease, particular colour of flower
- if original plant had unusual combination of characteristics due to selective breeding pr genetic modification, then this combination. van be retained without risk of losing that combination through sexual reproduction
- new plants are all uniform in their phenotype- makes them easier to grow/harvest
- using the apical bid (meristem) as an explant for tissue culture ensures the new plant are free from viruses
29
Disadvantages of artificial cloning in plants
- tissue culture is labour intensive
- expensive to set up facilities to perform tissue culture successfully
- tissue culture can fail due to microbial contamination
- all of the cloned offspring are genetically identical- therefore susceptible to the same pests/diseases- crops grown in monocultures allow rapid spread of a disease or pest between the closely planted crop plants
- no genetic variation, except that introduced by mutation
30
Briefly outline the nature of artificial cloning in animals
- natural cloning is rare in many species (except some invertebrates)- mean most cloning is artificial
- Strats with totipotent cells- only true ones are very early embryo cells
31
name 2 different categories of artificial cloning in animals
- reproductive cloning
- non-reproductive cloning
32
Describe reproductive cloning
- can produce large numbers of genetically identical animals
Can be useful for:
- elite farm animals produced by selective breeding (artificial selection) or geeing modification- e.g. particularly good individual bull whose value is as a stud (supplying sperm for artificial insemination)
- genetically modified animals developed with unusual characteristics e.g. stats that produce spider silk in their milk and cows that produce less methane
33
What are the 2 main techniques to achieve reproductive cloning
- embryo twinning
- somatic cell nuclear transfer (SCNT)
34
Outline the basis of embryo twinning
- mammals can produce identical offspring (twins) if an embryo splits in early development- has given rise to an artificial technique that has been used since the 1970s
- has been sued to clone elite farm animals or animals for scientific research
35
Steps of embryo splitting
1) a zygote (fertilised egg) is created by in Vito fertilisation (IVF)
2) The zygote is allowed to divide by mitosis to form a small ball of cells
3) The cells are separated and allowed to continue dividing
4) Each small mass of cells is placed into the uterus of a surrogate mother
36
Describe the outcome of embryo splitting
- precise genotype and phenotype of the offspring produced will depend on the sperm and egg used
- means precise phenotype will be unknown until the animals are born
37
Online the basis of somatic cell nuclear transfer
- only way to clone an adult
- phenotype down before cloning starts
- first performed on mammal in 1996- dolly the sheep
38
Steps of somatic cell nuclear transfer (SCNT)
1) Egg cell is obtained and its nucleus is removed- enucleation
2) A normal body cell (somatic cell) from the adult to be cloned is isolated and may have the nucleus removed
3) The complete adult somatic cell or its nucleus is fused with the empty egg cell by applying an electric shock
4) The shock also triggers the egg cell to start developing, as though it had just been fertilised
5) The cell undergoes mitosis to produce a small ball of cells
6) The young embryo is placed onto the uterus of a surrogate mother
39
Name different types of non-reproductive cloning
- therapeutic cloning
- cloning for scientific research
40
Describe therapeutic cloning
New tissues/organs can be grown as a replacement parts for people who are unwell:
- skin can be grown in vitro to act as graft over burned areas
- cloned ells have been used tor repair damage o the spinal cord of a mouse and to restore the capability to produce insulin in the pancreas
- potential to grow whole new organs to replace diseased organs
Tissue grown from the patents own cells will be genetically identical so avoid rejection- problem when transplanting donated organs
41
Describe cloning for scientific research
- genetically identical embryos can be used for scientific research into the action of genes that control development and differentiation
- can also be used to grow specific tosses organs for use in tests on the effects of medicinal drugs
42
Advantages of artificial cloning in animals
- can produce a hole herd of animals with a high yield or showing an unusual combination of characteristics e.g. producing silk in milk
- produces genetically identical copies of very high value individuals retaining the same characteristics
- using genetically identical embryos and tissues for scientific reproach allows the effects of genes and hormones to be assessed with no interference from different genotypes
- testing medicinal drugs on cloned cells/tissues avoids using animals or people for testing
- can produce cells and tissues genetically identical to the donor, for use in repairing damage caused by disease or accidents
- individuals from an endangered species can be cloned to increase numbers
43
Disadvantages of artificial cloning in animals
- lack of genetic variation may expose herd to certain diseases/pests
- animals may be produced with little regard for their welfare which may have undesirable side effects e.g. meat-producing chickens that can't walk
- success rate of adult cell cloning is very poor
- method is a lot more expensive than conventional breeding
- cloned animals may be less healthy and have shorter life spans e.g. dolly the sheep suffered from arthritis/chest tumours- aged prematurely
- ethical issues regarding how long the embryo survives and whether its right to create a life simply to destroy it
- increasing numbers of an endangered species founts help to increase genetic diversity
44
Describe the history of biotechnology
- name- Karoly Erkley- 1919- agricultural engineer who stet up industrialised farming unit- used term to describe any technological process that made use f living organism or pars of living organisms to manufacture useful products/provide useful services- included domestication of animals, panting of crops, mechanisation of agricultural processes and selective breeding
- oldest documented exmaple- production of beer.ale 7000 years ag- makes used of yeast to ferment maltose sugars in germinating barley, other early examples include making yoghurt, cheese, baking
- use of bacteria to produce acetone to make explosives in world war I
- manufacture of penicillin from fungus penicillin notatum in World War II
- soft of emphasis away from food towards manufacture of drugs
- new science of DNA technology brought biotechnology to current position- inctreasedunderstanding of genetics/genetic engineering along with ability to manipulate living conditions of living organisms has led to huge expansion In biotechnology- biggest expansion is in use of microorganisms in industrial processes
45
name the 4 main areas in which microorganisms are used in biotechnology
- food
- pharmaceutical drugs
- enzymes
- other products
46
Describe examples of microorganisms used in food (biotechnology)
- ethanol in beer and wine, carbon dioxide used to make bread rise- use yeast
- lactic acid used to make yoghurt and cheese - uses bacteria
- mycoprotein (filamentous fungus protein used to make vegetarian food- uses fungus
- soya- soya beans are fermented to produce soy sauce- uses yeast pr aspergillus
47
Describe examples of microorganisms used in pharmaceutical drugs (biotechnology)
- penicillin- uses penicillium fungus
- other antibiotic- ise other fungi and bacteria (mainly bactreia or genus sterptomyces)
- insulin and other theraptpeuric human proteins- genetcially modified bacetria
48
Describe examples of microorganisms used in enzymes (biotechnology)
* Protease and lipase used in washing powders- uses bacteria
*Pectinase used to extract juice from fruit- uses aspergillum
* Sucrase used to digest sugar to make food sweeter- yeasts an daspergillus
* Amylase to digest starch into sugar to produce syrup used as a sweetener in food production- uses A. oryzae
*Protease used to tenderise meat- aspergillus spp
* Lactase to make lactose-free milk- A.oryzae
* Removing sticky residues from recycled paper
49
Describe examples of microorganisms used in other products (biotechnology)
- biogas (combination of CO2 and methane)- uses anaerobic bacteria (decomposers)
- citric acid (find preservative)- fungus
- Bioremediation- cleaning waste water- variety of bacteria and fungi
50
Advantages of using microorganisms in biotechnology
- relatively cheap and easy to grow
- production process takes place at lower temperature than would be required to make the molecules by chemical engineering means- saves fuel and reduces costs
- production process can take place at normal atmospheric pressure- safer than using chemical reactions that may require very high pressure for successful manufacture of certain molecules
- the production process isn't dependent on climate- can take place anywhere in world with resources to build and run suitable equipment
- microorganism can be fed by-products from other food industries e.g. starch, waste water or molasses ( however often have to be pre-treated which can add to costs
- microorganisms have a short life cycle and reproduce quickly- some may reproduce as often as every 30 minutes under idea conditions means large population can grow very quickly indeed the reaction vessel (fermenter)
- microorganisms can be genetically modified relatively easily- allows very specific production process to be achieved
- fewer ethical considerations to worry about in using micro-organisms
- the products are often released form the microorganism into the surrounding medium- makes the product easy to harvest
- the product is often more pure or easier to isolate than in conventional chemical engineering process- means lower downstream processing costs
51
Describe the use of other organisms in biotechnology
- genetically modified mammals e.g sheep, goats and cows can be used to produce useful proteins
- in some mammals the proteins are incorporated into the milk and can be easily harvested e.g. goats that have been genetically modified to possess the gene for spider silk secrete it into their milk
- In other cases the protein may be secreted into the blood e.g. cows have been genetically modified to synthesise human antibodies, which can be isolated from their blood
52
What else can biotechnology mean (other than using organisms in the production process)
*gene technology
* genetic modification and gene therapy
* selective breeding
* cloning by embryo-splitting and micropropagation
*the use of enzymes in industrial processes
* immunology
53
Outline the use of microorganisms in the production of yoghurt
- yoghurt is milk that has undergone fermentation by Lactobacillus bulgaricus and streptoccus thermophilus
- the bacteria convert lactose to lactic acid
- the acidity denatures the milk protein, causing it to coagulate
- the bacteria partially digest the milk, making it easy to digest
- fermentation also produces the flavours characteristic of yoghurt
- other bacteria e.g. L. acidophilus, L. subsp. casei and Bifidobacterium may be added as probiotics- bacteria which may benefit human health by improving digestion of lactose, aiding gastrointestinal function and stimulating the immune system
54
Describe the use of microorganisms to make cheese
- milk is usually pre-treated with a culture off bacteria (lactobacillus) that can produce lactic acid from the lactose
- once it is acidified, the milk os mixed with rennet
- rennet contains the enzyme rennin (chymosin) which is found in the stomach of young mammals
- rennin coagulates the milk protein (casein) in the presence of calcium ions
- the resulting solid (curd) is separated from the liquid component (whey) by cutting, stirring and heating
- the bacteria continue to grow, producing more lactic acid
- the curd is then pressed into moulds
- treatment while making nd pressing the curd determines the characteristics of the cheese e.g. flavour determined during later ripening/maturing process, can be given additional flavour by inoculation with fungi e.g. penicillin to produce blue cheese
55
Describe the coagulation of casein in cheese production
1) kappa-casein, which keeps the casein in solution, os brown down- makes the casein insoluble
2) the casein is precipitated by the action of calcium ions, which bind the molecules together
56
Describe the use of microorganisms in baking (bread)
- bread is mixture of flower, water and slat with some yeast (single celled fungus, sacharomyces cerevisae)
1) Mixing- ingredients are mixed together thoroughly by kneading- produces dough
2) Proving/fermenting - dough is left in warm place for up to three hours while the yeast respires anaerobically- produces carbon dioxide bubbles, causing the dough to rise
3) Cooking - the risen dough is baked- any alcohol evaporates during the cooking process
57
Name foods which use microorganisms in tenor production
- yoghurt
- cheese
- bread baking
- alcoholic beverages
- single-cell protein
58
Describe the use of microorganisms in making alcoholic beverages
- product of anaerobic respiration of yeast (S.cerevisiae)
- Wine is made using grapes that naturally have yeasts on their skin, grapes contain the sugars fructose and glucose- when they are crushed, the yeasts uses these sugars to produce carbon dioxide and alcohol
- Ale/Beer is brewed using barley grains that ate beginning to germinate- malting- as the grain germinates, it converts stored starch to maltose, which is respired by the yeast- anaerobic respiration produces carbon dioxide and alcohol. Hops are used to give bitter taste to liquid
59
Describe the use of microorganism in the production of single-cell protein (SCP)
- microorganismnused most frequently is the fungus Fusarium venenatum
- The fungal protein or mycoprotein is also known as single-cell protein (SCP).
- e.g. Quorn- first produced in the early 1980s- marketed as a meat substitute for vegetarians and a healthy option for non-vegetarians, as it contains no animal fat or cholesterol
- huge potential in SCP production using such microorganisms as Kluyveromyces, Scytalidium and Candida- these fungi can produce protein with a 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 (curdled milk from which the curds have been removed)
60
Advantages of using micro-organisms in food production
- Production of protein can be many times 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
* The microorganisms provide a good source of protein.
* The protein contains no animal fat or cholesterol.
*The microorganisms can easily be genetically modified to adjust the amino acid content of the protein
* SCP production could be combined with removal of waste products
* Production is independent of seasonal variations
* Not much land required
61
Disadvantages of using micro-organisms in food production
* Some may not want to eat fungal protein/food that has been grown on waste
* Isolation of the protein - the microorganisms are grown in huge fermenters and need to be isolated from the material on which they grow
* The protein has to be purified to ensure it is uncontaminated
* Microbial biomass can have a high proportion of nucleic acids, which must be removed.
* The amino acid profile may be different from traditional animal protein e.g. can be deficient in methionine.
* Infection - conditions needed for the microorganisms to grow are also ideal for pathogenic organisms- care must be taken to ensure the culture is not infected with the wrong organisms.
- Palatability - protein doesn't have the taste or texture of traditional protein sources
62
What does commercial drug production use
fermenters
63
describe fermenters used in commercial drug production
- large stainless steel container
- growing conditions can be controlled to ensure the best possible yield of the product
64
Describe the conditions that must be controlled in scaling up production of drugs and why
- temperature- enzymes will denature if too hot but growth will be limited if too cold
- nutrients available- microorganisms require nutrients to grow and synthesise the product- sources of carbon, nitrogen, minerals, and vitamins are needed
- oxygen availability- most microorganisms res[ire aerobically
- pH- enzyme activity and hence growth and synthesis are affected by extremes of pH
- concentration of product- if the product is allowed to build up, synthesis process may be affected
64
Describe the conditions that must be controlled in scaling up production of drugs and why
- temperature- enzymes will denature if too hot but growth will be limited if too cold
- nutrients available- microorganisms require nutrients to grow and synthesise the product- sources of carbon, nitrogen, minerals, and vitamins are needed
- oxygen availability- most microorganisms res[ire aerobically
- pH- enzyme activity and hence growth and synthesis are affected by extremes of pH
- concentration of product- if the product is allowed to build up, synthesis process may be affected
65
Describe the process of scaling up drug production
- fermenter must be sterilised using superheated stream
- it can then be filed with all the components required for growth and supplied with a starter culture of microorganism to be used
- the culture will be left to grow and synthesise the products
66
Describe continuous culture (scaling up drug production)
- some products are synthesised by the microorganism during normal metabolism when they are actively growing- primary metabolites
- such products continuously released from the cells and cam be extracted continuously from the fermenting broth
- the broth is topped up with nutrients as these are used by the microorganisms
- some of the broth is removed regularly top extract the product and remove cells from the broth- otherwise population becomes too dense
- continuous culture keeps microorganisms growing at a specific growth rate
67
Describe batch culture (scaling up drug production)
- products are produced only when cells are placed under stress e.g. high population density/limited new nutrient availability- called secondary metabolites
- produced mostly during stationary phase of growth
- culture set up with limited quantity of nutrients and allowed to ferment for a specific time
- after this time, the fermenter is empties and the product can be extracted from the culture
68
What is asepsis in scaling up drug production
- Ensuring sterile conditions are maintained
- in products where foods or medicinal chemicals are produced, all products must be discarded if contamination by unwanted organisms occur
69
Why is asepsis important in scaling up drug production
The nutrient medium used would also support the growth of unwanted microorganisms- wood reduce population because the unwanted microorganisms:
- compete with he cultured microorganisms for nutrients and space
- reduce the yield for useful products
- spoil the product
They may also:
- produce toxic chemicals
- destroy the cultured microorganisms and their products
70
name an example of when a drug has been successfully Mass-produced
Penicillin
71
Online the mass-production of penicillin
- Florey and Chain
- fermentation by the fungus penicillium chrysogenum
- modern strains of the fungus have been selectively bred to be more productive than the early strains
- penicillin is a secondary metabolite- only produced once the population has reached a certain size- therefore manufactured by batch culture
72
Describe the process of the mass-production of penicillin
1) Fermenter run for 6-8 days
2) Culture Is then filtered to remove the cells
3) The antibiotic is precipitated as crystals by the addition of potassium compounds0 the antibiotic may be modified by the action of other microorganisms or by chemical means
4) The antibiotic is mixed with inert substances and prepared for administration in tablet form, as a syrup or in a form suitable for injection
Batch culture- secondary metabolite
73
Describe the development in the production of insulin (biotechnology)
- was previously extracted from pancreas of animals e.g. pigs/cattle sent for slaughter, however, insulin from slaughtered animals is not identical to human insulin and so is less effective than human insulin, and expensive to extract
- In 1978, synthetic human insulin was developed by genetically modifying a bacterium- gene for human insulin was combined with a plasmid to act as a vector, so the gene could be inserted into the bacterium Escherichia coli
- The resulting genetically-modified bacterium enabled the production of vast quantities of human insulin at relatively low cost
- Manufactured by continuous culture- primary metabolite
74
Define biotechnology
The use of living organisms or parts of living organisms in industrial processes
75
What is bioremediation
- the use of microorganisms to clean the soil and underground water on polluted sites
- the organisms convert the toxic pollutants o less harmful substances
- idea started when pseudomonas bacteria was modified- enabling it to break down crude oil- proposed it could be used in treating oil spills
- solvents and pesticides can also be treated using bioremediation
76
Describe the process of bioremediation
- invokes stimulating the growth of suitable microbes that se the contaminants as a source of food
- requires the right conditions for the growth of microorganisms- available water, suitable temperature, suitable pH
77
Describe what happens if the conditions for bioremediation aren't suitable
- Where conditions are not quite suitable, they may be modified by the addition of suitable substances
- In some cases, additional nutrients e.g. molasses may be needed to ensure the microorganisms can grow effectively
- May also be necessary to pump in oxygen for aerobic bacteria
- Where conditions cannot be made suitable in situ, the soil may be dug up and moved to be treated ex situ
78
What are advantages of bioremediation
* Uses natural systems
* Less labour/equipment required
* Treatment in situ
* Few waste products
* Less risk of exposure to clean-up personnel
79
What are disadvantages of bioremediation
Only suitable for certain products- heavy metals e.g. cadmium and lead can't be treated
80
Name different ways of growing organisms in the laboratory
Types of growth medium:
- Broth- soup-like liquid- kept in bottles or tubes
- Agar- set jelly-like substance- melted and poured into Petri dishes
81
What do microrgansisms need to grow
Will grow on any material that provides the carbon compounds for respiration and a source of nitrogen for protein synthesis
82
What does typical nutrient agar contain
- peptones (from the enzymatic breakdown of gelatine)
- yeast extract
- salts and water
- may also contain glucose or blood
83
Why have aseptic techniques been developed
- to reduce the likelihood of contaminating the medium with unwanted bacteria or fungi
84
Describe the standard procedure of ascetic techniques
1) Wash hands
2) Disinfect working area
3) Have Bunsen burner operating nearby to heat air- causes air to rise and prevents airborne microorganisms settling, creates area around iy of sterile air in which microbiologist can work
4) As vessel is opened, pass the neck of the bottle over the flame to prevent bacteria ion air entering the bottle;e, bottle should be flames as it is closed
5) Don't live lid off Petri dish completely- just open enough to allow introduction desired microorganism
6) Any glassware/metal equipment should be passed through flame before and after contact with the desired microorganisms
85
What are the main steps of (growing microorganisms on agar plates)
1) sterilisation
2) inoculation
3) incubation
86
Describe sterilisation (growing microorganisms on agar plates)
- nutrient agar medium and any equipment must be sterilised
- medium- sterilised by heating an autoclave at 121oC for 15 mins (high temperature achieved by boiling water under high pressure inside the autoclave)- kills all living organisms including any bacteria/fungal spores
- when medium has cooled enough to handle, pour int sterile petri dish and leave to set
- keep lid on Petri dish- prevent infection
- all equipment sterilised from this point onwards
87
What is inoculation
The introduction of microorganisms ti a sterile medium
88
describe different inoculation techniques
*Streaking -wire inoculating loop is used to transfer a drop of liquid medium onto the surface of the agar. The drop is drawn out into a streak by dragging the loop across the surface. Need to be careful not to break the surface of the agar.
* Seeding - sterile pipette used to transfer small drop of liquid medium to surface of agar or to the Petri dish before agar is poured in
*Spreading - sterile glass spreader used to spread the inoculated drop over the surface of the 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 the agar medium
89
Describe the incubation of Petri dishes
- must be labelled
- top should be taped to bottom using 2 strips of adhesive tape- shouldn't seal Petri dish completely as can lead too election of anaerobic bacteria- may be pathogenic
- Petri dish placed in a suitable warm environment e.g. incubator
- should be placed upside down- prevents drops of condensation falling onto surface of agar, prevents agar medium drying out too quickly
- suitable temperatures depend on organism being grown
- cultures can be examined after 24-26 hours- don't open Petri dish
90
Describe different colonies grown on agar plates
- bacteria grown into visible colonies- each colony results from single bacterium
- colonies may be shiny or dull
- some cookies are round with entire edges, others have crenated edges
- colonies can be range of different colours
- filamentous fungi grow into a mass of hyphae- may also be circular, but mass is not shiny and often looks like cotton will with fluffy aerial hyphae
- single-celled fungi grow as circular colonies
91
Describe the disposal of Petri dishes
- 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 sit could be source of infection
92
Describe the use of a liquid medium for growing microorganism cultures
- initially clear but will turn cloudy when bacteria have grown
- can be useful to increase the numbers of microorganisms before transferring ti agar plates for counting or identification
- similar aseptic techniques must be applied
-b can be sed to investigate population growth
93
Outline how population growth in a closed culture is investigated
- liquid bright can be used to measure the growth rate of a. microorganism population
- A sterile growth is inoculated and the population size is measured at regular intervals during incubation
- the population size can be measured by transferring a small sample to an agar plate and incubating the agar culture- each individual microorganism will produce a visible colony
94
What can be an issue with measuring population growth in a closed culture, what is done to avoid this
- the number of individual organisms in bright can be high
- if broth used to inoculate agar plate, ether may be too many colonies which merge together making a count impossible
- in order to investigate rate of growth need to reduce population density- can be achieved by serial dilution
95
Describe how to do a serial dilution of broth culture
- step-wise dilution of the broth culture
- At each step the broth is diluted by a factor of 10
- Take a 1 cm3 sample of the broth and add 9 cm3 of distilled water - label the diluted broth as '10-1'
- Take a 1 cm3 sample of this diluted broth and add 9 cm° of distilled water - label as '10-2'
- Continue this procedure until you have a series of dilutions with suitable labels
- A drop of each dilution can be used to inoculate an agar plate- one will produce a culture plate in which the number of colonies can be counted
- When recording the population density
- do not forget to multiply your count by the dilution factor and also by the volume added to the plate
96
What is a closed growth culture
- A population in which all the conditions are set at the start and there is no exchange with the external environment
97
Describe what will happen to a small population of microorganisms in a closed culture that contains all the nutrients required for growth
- will undergo population growth which will follow a predictable pattern- lag phase, log (exponential) phase, stationary phase, and death (decline) phase
98
Describe the lag phase (microorganism growth in closed culture)
Population shiest grow quickly- population is small and adjusting to new environment including:
- taking up water
- cell growth
- switching on (activating) certain genes
- synthesising specific proteins (enzymes)
99
Describe the log (exponential) phase (microorganism growth in closed culture)
- organism has adjusted to their environment
- they each have the enzymes needed to survive
- each individual has sufficient nutrients and space to grow rapidly and reproduce
- the population doubles in size with each generation
- on some microorganisms, this cam be as frequently as once every 20-30 minutes
100
Describe the stationary phase (microorganism growth in closed culture)
- eventually the increasing number of organisms use up the nutrients and produces increasing amounts of waste products e.g. CO2 and other metabolites
- the rate of population growth declines and the number of individuals ding decreases until the reproduction rate equals the death rate
- no population growth
101
Describe the death (decline) phase (microorganism growth in closed culture)
- the nutrients run out and the concentration of waste products may become lethal
- more individual die than are produced and large population begins to fall
- eventually all the organisms will die
102
Describe the production of primary and secondary metabolites in closed culture microorganism population growth
- primary metabolites produced during the normal activities of he microorganism will be collected from a fermenter during the log phase
- in a fermenter, the population is not kept in a closed culture, but conditions are maintained for optimal growth
- secondary metabolites are produced in the stationary phase- population must be kept in a closed culture and the metabolites can eb collected at the end of the stationary phase or during the decline phase
103
What is an immobilised enzyme
An enzyme that is held in place and not free to diffuse through the solution- taken out of suspension and held so they don't freely mix with the substrate
104
Briefly outline use of immobilised enzymes in biotechnology
- enzymes aren't used up in teh reaction and remain in suspension when reaction has been completed
- in industrial process, means product must be isolated from the enzymes before use- could be expensive
- means process can be simplified by taking the enzymes out of the microorganisms
105
What are the advantages of the fact that immobilised enzymes don't mix freely with the substrate
- extraction costs lower and enzyme and product don't mix
- enzymes can easily be reused
- a continuous process is made easier as there are no cells requiring nutrients, reproducing, and releasing waste products
- the enzymes are surrounded by the immobilising matrix, which protects them from extreme conditions- can use higher temperatures or a wider pH range without denaturing
106
Name different methods of immobilising enzymes
- adsorption
- covalent bonding
- entrapment
- membrane separation
107
Describe adsorption as a method of immobilising enzymes
- enzyme molecules are bound to a supporting surface by a combination of hydrophobic interactions and ionic links
- suitable surfaces- clay, porous carbon, glass beads, resins
- the enzyme molecules are bound with the active site exposed and accessible to the substate, however, the active site may be slightly distorted by the additional interactions affecting enzyme activity
108
Issues with adsorption as a method of immobilising enzymes
- the active site may be slightly distorted by the additional interactions affecting enzyme activity
- the bonding faces are not always strong, and enzymes can become detached and leak into the reaction mixture
109
Describe covalent bonding as a method of immobilising enzymes
- enzyme molecules are bonded to a supporting surface such as clay using strong covalent bonds
- bonded using a cross-linking agent, which may also link them in a chain
110
Advantages and disadvantages of covalent bonding as a method of immobilising enzymes
- production of covalent bonding ca be expensive and ca distort the enzyme active site, reducing activity
- however the enzymes are much less likely to become detached and leak into the reaction mixture
111
Describe entrapment as a method of immobilising enzymes
- enzyme molecules are trappend in a matrix that doesn't allow free movement
- the enzyme molecules are unaffected by entrapment and remain fully active
- the substrate molecules must diffuse into the entrapment matrix, and the product molecules must be able to diffuse out- therefore suitable only for processes where the substrate and product molecules are relatively small
- calcium alginate beads often used in schools, industrial processes may use a cellulose mesh
112
Describe membrane separation as a method of immobilising enzymes
- enzyme molecules are separated form the reaction mixture by a partially permeable membrane
- substrate and product molecules must be small enough to pass through the partially permeable membrane by diffusion
113
Issues with membrane separation as a method of immobilising enzymes
substrate and product molecule must pas through partially membrane by diffusion- access to enzymes may limit the reaction rate
114
name different industrial uses of immobilised enzymes
- glucose isomerase (AKA xylose isomerase)
- penicillin acyclase (amidase)
- lactase
- aminoacyclase
- glucoamylase
- nitrile hydratase
115
Describe glucose isomerase (AKA xylose isomerase)
- converts glucose to fructose- most widely used as number of applications of syrup produced
- used to produce high fructose corn syrup (HFCS)
- much sweeter than sucrose- use in 'diet foods' as less sugar needed for equivalent sweetness, may be used in sweeter foods for diabetics
- chapter than sucrose- used to replace in food industry e.g. soft drinks, cereals, ham, ice-cream, yoghurt, sliced ham
116
Describe penicillin acyclase (penicillin amidase)
- formation of semi-syntehtic penicillins e.g. amoxicillin and ampicillin-were first developed during 1960s
- some penicillin-resistant microorganisms aren't resistant to these semi-synthetic penicillins
117
Describe lactase (immobilised enzymes)
- converts lactose to glucose and galactose by hydrolysis
- used to produce lactose-free milk
- milk important source of calcium- strong bones and teeth- people with insufficient calcium in diet more likely to develop weak bones or osteoporosis
- therefore important that people who are lactose intolerant (unable to digest and absorb lactose in milk) are given lactose-free milk or calcium supplements
118
Describe aminoacyclase
- hydrolase used to produce pure samples of L-amino acids by removing the acyl group from the nitrogen of an N-acyl-amino acid
- L-amino acids are used as the building blocks for synthesis of a number of pharmaceutical and agrochemical compounds, may also be used as additives for human food and animal feedstuffs
119
Describe glucoamylase
- converts dextrins to glucose
- during hydrolysis of starch, short polymers of glucose (dextrins) are fomed- hydrolysis by glucoamylase can convert these dextrins to glucose
- glucoamylase can be immobilised on a variety of surfaces and used to digest sources of starch such as corn and cassava
- the enzyme is used in a wide range of fermentation processes, including the conversion of a starch pulp to alcohol used to produce gasohol- an alternative fuel for motor veichles
- also used in the food industry to able high fructose corn syrup
