Cloning and Biotechnology M6 C22

Natural cloning in plants
_______ _________ forms a structure which develops into a fully differentiated new plant, which is genetically identitical to the parent. The new plant may be propogated from the ____, leaf, ___ or root of the parent plant.

Vegetative propagation forms a structure which develops into a fully differentiated new plant, which is genetically identitical to the parent. The new plant may be propogated from the stem, leaf, bud or root of the parent plant.

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Vegetative propagation involvs perennating ______, enabling the plant to survive in adverse conditions. These contained stored food from __________ and remain ______ in the soil. What are the 4 places in a plant where natural cloning occurs?

Vegetative propagation involvs perennating organs, enabling the plant to survive in adverse conditions. These contained stored food from photosynthesis and remain dormant in the soil. Bulbs, runners, rhizomes, stem tubers

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Bulbs
eg. daffodil The leaf bases ____ with stored food from photosynthesis. Buds form internally which develop into new ______ and new plants in the next growing season.

eg. daffodil The leaf bases swell with stored food from photosynthesis. Buds form internally which develop into new shoots and new plants in the next growing season.

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Runners
eg. strawberry and spider plant. A lateral ____ grows away from the parent plant and roots develop where the runner touches the ground. A new plant develops, the runner eventually withers away leaving the new individual _________

eg. strawberry and spider plant. A lateral stem grows away from the parent plant and roots develop where the runner touches the ground. A new plant develops, the runner eventually withers away leaving the new individual independent

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Rhizomes
eg. maram grass. A rhizome is a specialised horizontal ____ running underground, often _____ with stored food. Buds develop and form new vertical _____ which become independent plants.

eg. maram grass. A rhizome is a specialised horizontal stem running underground, often swollen with stored food. Buds develop and form new vertical shoots which become independent plants.

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Stem tubers
eg. potato. The tip of an underground stem becomes ______ with stored food, to form a storage organ. ____ on the storage organ develop to produce new shoots

eg. potato. The tip of an underground stem becomes swollen with stored food, to form a storage organ. Buds on the storage organ develop to produce new shoots

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Use of natural clones in horticulture:
Splitting up bulbs, removing young plants from runners, cutting up rhizomes all increase plant numbers _______ and the new plants have the same genetic ___________ as the parents.

Splitting up bulbs, removing young plants from runners, cutting up rhizomes all increase plant numbers cheaply and the new plants have the same genetic characteristics as the parents.

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Propogation from cuttings is ______ than using seeds, and the ______ of plants is guaranteed. By taking cuttings from good stock, the offspring will be genetically identical and therefore ____ well.

Propogation from cuttings is faster than using seeds, and the quality of plants is guaranteed. By taking cuttings from good stock, the offspring will be genetically identical and therefore crop well.

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However there will be a lack of genetic variation in the offspring, so there's a risk when a new _______ appears, or ____ appear, or climate change occurs.

However there will be a lack of genetic variation in the offspring, so there's a risk when a new disease appears, or pests appear, or climate change occurs.

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How to take plant cuttings:
1. use a non-________ stem
2. make a cut into the stem
3. use _______ rooting powder
4. reduce leaves to _ or _

1. use a non-flowering stem
2. make a cut into the stem
3. use hormone rooting powder
4. reduce leaves to 2 or 4

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5. keep cutting well _______
6. cover the cutting with a plastic ___ for a few days

5. keep cutting well watered
6. cover the cutting with a plastic bag for a few days

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Micropropagation using tissue culture (artificial cloning):
Makes large numbers of genetically _______ offspring from a single parent plant. Used when a desirable plant doesn't produce ____, doesn't respond well to _____ cloning, is ____, has been genetic

Makes large numbers of genetically indentical offspring from a single parent plant. Used when a desirable plant doesn't produce seeds, doesn't respond well to natural cloning, is rare, has been genetically modified, needs to be pathogen free

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1. take a small sample of tissue from the plant you want to clone. ________ tissue and ____ often dissected out in sterile conditions. This avoids ___________ by fungi and bacteria, tissue is also usually virus-free.

1. take a small sample of tissue from the plant you want to clone. Meristem tissue and buds often dissected out in sterile conditions. This avoids contamination by fungi and bacteria, tissue is also usually virus-free.

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2. The sample is sterilised, usually immersed in ______ or ______. The latter doesn't need to be rinsed off, which means the tissue is more likely to remain _____. The material removed from the plant is called the _______.

2. The sample is sterilised, usually immersed in bleach or ethanol. The latter doesn't need to be rinsed off, which means the tissue is more likely to remain sterile. The material removed from the plant is called the explant.

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3. The explant is placed in sterile culture medium containing plant hormones such as _____ and ________, stimulating mitosis. The cells multiply, forming a mass of identical cells knows as ______.

3. The explant is placed in sterile culture medium containing plant hormones such as auxins and cytokinins, stimulating mitosis. The cells multiply, forming a mass of identical cells knows as callus.

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4. The callus is _______ up and individual cells are transferred to a new culture medium containing a different mixture of hormones, stimulating the development of tiny genetically identical _______.

The callus is divided up and individual cells are transferred to a new culture medium containing a different mixture of hormones, stimulating the development of tiny genetically identical plantlets.

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5. Planted are potted into ______ where they grow into small plants
6. Young plants are planted out to grow and produce a ____.

5. Planted are potted into compost where they grow into small plants
6. Young plants are planted out to grow and produce a crop.

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Advantages of micropropagation:
- Allows for the ____ production of a large number of plants with known genetic make-up, which will yield good ____.
- Culturing meristem tissue produces ______-free plants.

- Allows for the rapid production of a large number of plants with known genetic make-up, which will yield good crops.
- Culturing meristem tissue produces disease-free plants.

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- Makes it possible to produce viable numbers of plants after genetic __________ of plant cells
- Produces large numbers of plants which are seedless and therefore _____ to meet consumers tastes

- Makes it possible to produce viable numbers of plants after genetic modification of plant cells
- Produces large numbers of plants which are seedless and therefore sterile to meet consumers tastes

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- Grows plants which are naturally ______ or difficult to grow from seed
- Reliably increases the numbers of _________ plants

- Grows plants which are naturally infertile or difficult to grow from seed
- Reliably increases the numbers of endangered plants

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Disadvantages of micropropagation:
- Produces a ________, where many plants are genetically identical, so all susceptible to the same _______
- Relatively _______ and requires skilled workers

- Produces a monoculture, where many plants are genetically identical, so all susceptible to the same _______
- Relatively expensive and requires skilled workers

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- Explants and plantlets vulnerable to _______ by moulds and other diseases during production
- If source material is _______, all of the clones will be too
- Large numbers of new plants are ____ during the process

- Explants and plantlets vulnerable to infection by moulds and other diseases during production
- If source material is infected, all of the clones will be too
- Large numbers of new plants are lost during the process

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Natural cloning in animals (invertebrates):
Starfish can _______ animals from fragments of the original if they are damaged. Flatworms and sponges form new identical _____ as part of their reproductive process

Starfish can regenerate animals from fragments of the original if they are damaged. Flatworms and sponges form new identical clones as part of their reproductive process

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Hydra produce small ____ on the side of their body which develop into genetically identical clones. In some insects, females can produce offspring without ______.

Hydra produce small buds on the side of their body which develop into genetically identical clones. In some insects, females can produce offspring without mating.

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Cloning in animals (vertebrates):
Forming ________ (identical) twins. Early embryo splits to form 2 separate embryos, not sure the trigger of this. The ________ at which twins occur varies between species.

Forming monozygotic (identical) twins. Early embryo splits to form 2 separate embryos, not sure the trigger of this. The frequency at which twins occur varies between species.

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When identical twins are born, although genetically identical, they may look different as a result of differences in their _______ and _______ in the uterus.

When identical twins are born, although genetically identical, they may look different as a result of differences in their position and nutrition in the uterus.

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Artificial cloning (artificial twinning in cattle):
1. Cow with desirable traits treated with hormones so she ____-______, releasing more mature ___ than normal

1. Cow with desirable traits treated with hormones so she super-ovulates, releasing more mature ova than normal

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2. Ova may be fertilised naturally, or by artificial _________ by a bull with good traits. The early embryos are gently flushed out the ______.
3. Alternatively, mature eggs removed and fertilised by top quality bull ____ in a lab

2. Ova may be fertilised naturally, or by artificial insemination by a bull with good traits. The early embryos are gently flushed out the uterus.
3. Alternatively, mature eggs removed and fertilised by top quality bull semen in a lab

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4. Before or around day 6, when cells are still _______, the cells of the early embryo are split to produce several smaller embryos, each capable of growing to form a healthy ____.

4. Before or around day 6, when cells are still totipotent, the cells of the early embryo are split to produce several smaller embryos, each capable of growing to form a healthy calf.

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5. Each of the split embryos is grown in the lab for a few days to ensure all is well before ________ into a surrogate mother. Each embryo goes into a different mother as single pregnancies have fewer _____ than twin pregnancies.

5. Each of the split embryos is grown in the lab for a few days to ensure all is well before implanted into a surrogate mother. Each embryo goes into a different mother as single pregnancies have fewer risks than twin pregnancies.

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6. Embryos develop into _______ and are born normally, a number of identical cloned animals are produced by different mothers.

6. Embryos develop into foetuses and are born normally, a number of identical cloned animals are produced by different mothers.

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Artificial cloning (somatic cell nuclear transfer):
1. ______ removed from a somatic cell of an adult animal
2. ______ removed from a mature ovum from a different female animal of the same species, it is _________.

1. Nucleus removed from a somatic cell of an adult animal
2. Nucleus removed from a mature ovum from a different female animal of the same species, it is enucleated.

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3. Nucleus from adult somatic cell placed into enucleated ovum, given a mild _____ ____ so it fuses and begins to divide. Sometimes the nucleus from the adult cell isn't removed but it placed next to the ovum, and the two cells fuse and divide under _____

3. Nucleus from adult somatic cell placed into enucleated ovum, given a mild electric shock so it fuses and begins to divide. Sometimes the nucleus from the adult cell isn't removed but it placed next to the ovum, and the two cells fuse and divide under e

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4. Embryo developed is transferred into the ______ of a third animal where it develops
5. New animal is a clone of the animal from which the original somatic cell was derived, although __________ DNA comes from egg cell.

4. Embryo developed is transferred into the uterus of a third animal where it develops
5. New animal is a clone of the animal from which the original somatic cell was derived, although mitochondrial DNA comes from egg cell.

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Advantages for animal cloning:
- artificial twinning enables high yielding farms to produce many more ______ than normal reproduction

- artificial twinning enables high yielding farms to produce many more offspring than normal reproduction

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- artificial twinning enables the success of a male animal at passing on desirable genes to be _________. If first cloned embryo resulted in a successful breeding animal, then more identical animals can be formed from the remaining _____ clones.

- artificial twinning enables the success of a male animal at passing on desirable genes to be determined. If first cloned embryo resulted in a successful breeding animal, then more identical animals can be formed from the remaining frozen clones.

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- SCNT enables GM embryos to be _________ and to develop, giving many embryos from one engineering procedure.
- SCNT enables cloning of ______ animals eg. cloning top-class race horses.

- SCNT enables GM embryos to be replicated and to develop, giving many embryos from one engineering procedure.
- SCNT enables cloning of specific animals eg. cloning top-class race horses.

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- SCNT has the potential to enable ________ or even _____ animals to be reproduced. The nucleus from dried or frozen tissue can be transferred to the egg of a similar living species and used to produces clones of a _____ species.

- SCNT has the potential to enable endangered or even extinct animals to be reproduced. The nucleus from dried or frozen tissue can be transferred to the egg of a similar living species and used to produces clones of a dead species.

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Disadvantages of animal cloning:
- SCNT is very ________, in most animals it takes many eggs to produce a single cloned offspring.
- many cloned animals fail to develop and _______ or produce ________ offspring.

- SCNT is very inefficient, in most animals it takes many eggs to produce a single cloned offspring.
- many cloned animals fail to develop and miscarry or produce malformed offspring.

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- many animals produced by cloning have shortened ________, although cloned mice have been developed to live their normal two years.

- many animals produced by cloning have shortened lifespans, although cloned mice have been developed to live their normal two years.

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Biotechnology:
applying biological organisms or enzymes to the ______, __________, or __________ of materials in the service of people.

applying biological organisms or enzymes to the synthesis, breakdown, or transformation of materials in the service of people.

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The uses of microorganisms in biotechnology:
- no _______ issues to consider, all is needed is the optimum conditions for growth.
- enormous range of microorganisms capable of carrying out different chemical _______ or ________ that can be used.

- no welfare issues to consider, all is needed is the optimum conditions for growth.
- enormous range of microorganisms capable of carrying out different chemical syntheses or degradations that can be used.

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- genetic engineering allows us to artificially _______ microorganisms to carry out synthesis reactions that they would not do naturally eg. produce human ______.

- genetic engineering allows us to artificially manipulate microorganisms to carry out synthesis reactions that they would not do naturally eg. produce human insulin.

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- microorganisms have a short ____ cycle and rapid _____ rate. So given the right conditions of food, oxygen and temperature, huge quantities of microorganisms can be produced in a _____ time.

- microorganisms have a short life cycle and rapid growth rate. So given the right conditions of food, oxygen and temperature, huge quantities of microorganisms can be produced in a short time.

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- conditions which most microorganisms need to grow include low ________, a supply of ______ and ____, and removal of waste gases. They provide their own catalysts, making bioprocesses relatively _____ compared to high temperatures and pressures needed in

- conditions which most microorganisms need to grow include low temperature a supply of oxygen and food, and removal of waste gases. They provide their own catalysts, making bioprocesses relatively cheap compared to high temperatures and pressures needed

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Advantages of using microorganisms to make food:
- microorganisms are more efficient than animals at converting energy into ______.
- microorganisms ______ quicker than plants and animals, so selective breeding is quicker.

- microorganisms are more efficient than animals at converting energy into biomass.
- microorganisms reproduce quicker than plants and animals, so selective breeding is quicker.

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- microorganisms can be grown on substrates that are _____ products of other industries eg. whey, a waste product from cheese-making

- microrganisms can be grown on substrates that are waste products of other industries eg. whey, a waste product from cheese-making

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Disadvantages of using microorganisms to make food:
- if conditions aren't ideal, microorganisms don't ____ properly, so don't work ________.

- if conditions aren't ideal, microorganisms don't grow properly, so don't work efficiently.

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- conditions that are ideal for microorganisms are also ideal for microorganisms that cause food to go off, or cause _______. So the processes have to be sterile, though this is _______.

- conditions that are ideal for microorganisms are also ideal for microorganisms that cause food to go off, or cause disease. So the processes have to be sterile, though this is expensive.

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- microorganisms used in food production have been genetically _________, and some people have ethical issues with the use of __ organisms so may not buy the product.

- microorganisms used in food production have been genetically modified and some people have ethical issues with the use of GM organisms so may not buy the product.

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Culturing microorganisms:
- need food as well as the right conditions of ______, ________, and __. This food provided is the ______ medium. It is either in liquid form (____) or solid form (____). Medium must be sterile until use using _____ techniques.

- need food as well as the right conditions of oxygen, temperature, and pH.. This food provided is the nutrient medium. It is either in liquid form (agar) or solid form (broth). Medium must be sterile until use using aseptic techniques.

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Inoculating broth (bacteria added):
1. make a _________ of the bacteria to be grown
2. mix a known volume with the sterile nurtient ____ in the flask

1. make a suspension of the bacteria to be grown
2. mix a known volume with the sterile nurtient broth in the flask

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3. stopper the flask with _____ ____ to prevent contamination from the air
4. incubate at a suitable ___________, shake regularly to provide ______ for the growing bacteria.

3. stopper the flask with cotton wool to prevent contamination from the air
4. incubate at a suitable temperature, shake regularly to provide oxygen for the growing bacteria.

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Inoculating agar (adding bacteria):
1. wire inoculating hoop _______ by holding it in a Bunsen flame until it glows red hot, it can't touch any surfaces as it cools to avoid __________.

1. wire inoculating hoop sterilised by holding it in a Bunsen flame until it glows red hot, it can't touch any surfaces as it cools to avoid contamination.

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2. Dip sterilised hoop into bacterial ________. Remove lid of the petri dish and make a ___-___ streak across the agar's surface. Avoid the loop digging into the agar by holding it almost _________. Surface of the agar must be kept ______.

2. Dip sterilised hoop into bacterial suspension. Remove lid of the petri dish and make a zig-zag streak across the agar's surface. Avoid the loop digging into the agar by holding it almost horizontal. Surface of the agar must be kept intact.

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3. Replace the lid of the petri dish, it should be held down with ____ but not sealed completely so ______ can get in, preventing the growth of anaerobic bacteria. ______ at a suitable temperature.

3. Replace the lid of the petri dish, it should be held down with tape but not sealed completely so oxygen can get in, preventing the growth of anaerobic bacteria. Incubate at a suitable temperature.

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What are the 2 main ways of growing microorganisms on an industrial scale?

Batch fermentation
Continuous fermentation

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Batch fermentation:
1. microorganisms inoculated in a fixed volume of ______
2. growth takes place, nutrients used up, both new ______ and _____ products build up.

1. microorganisms inoculated in a fixed volume of culture.
2. growth takes place, nutrients used up, both new biomass and waste products build up.

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3. culture reaches _________ phase, overall growth ceases, microorganisms carry out ________ changes to form the desired end products.

3. culture reaches stationary phase, overall growth ceases, microorganisms carry out biochemical changes to form the desired end products.

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4. process is stopped before _____ phase and the products are harvested. The whole system is then cleaned and ________, new batch culture started up.

4. process is stopped before death phase and the products are harvested. The whole system is then cleaned and sterilised, new batch culture started up.

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Continuous fermentation:
1. microorganisms inoculated into sterile _____ medium and start to grow
2. same medium is continually added to culture once it reaches the _________ phase.

1. microorganisms inoculated into sterile nutrient medium and start to grow
2. same medium is continually added to culture once it reaches the exponential phase.

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3. culture broth continually removed, containing the ______, _____ products, microorganisms, and the desired ______. This keeps the volume in the bioreactor constant.

3. culture broth continually removed, containing the medium, waste products, microorganisms, and the desired product. This keeps the volume in the bioreactor constant.

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Growth curve of a microorganism
What are the 4 stages to the growth curve?

1. Lag phase
2. Log/exponential phase
3. Stationary phase
4. Decline/death stage

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1. Lag phase
Numbers of bacteria is constant. Bacteria is ________ to their new environment. They are growing, synthesising the _______ they need, and aren't yet __________ at their maximum rate.

Numbers of bacteria is constant. Bacteria is adapting to their new environment. They are growing, synthesising the enzymes they need, and aren't yet reproducing at their maximum rate.

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2. Log/exponential phase
Numbers of bacteria increasing. Rate of bacterial ___________ is close to or at its theoretical maximum.

Numbers of bacteria increasing. Rate of bacterial reproduction is close to or at its theoretical maximum.

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3. Stationary phase
Numbers of bactera is constant again. The total growth rate is zero, the number of new cells formed by _____ ______ is cancelled out by the number of cells dying.

Numbers of bactera is constant again. The total growth rate is zero, the number of new cells formed by binary fission is cancelled out by the number of cells dying.

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4. Decline/death stage
Numbers of bacteria decreases. __________ has almost ceased and the _____ rate of cells is increasing.

Numbers of bacteria decreases. Reproduction has almost ceased and the death rate of cells is increasing.

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What is the formula for the number of individual organisms?

N = N(small 0) x 2^n

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What are the 5 factors affecting the growth rate of microorganisms?

Nutrients available
Oxygen levels
Temperature
Build-up of waste
Change in pH

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Nutrients available:
Initially plenty of food, but as number of microorganisms multiply ___________ it is used up. Nutrient level becomes insufficient to support further _____ and ___________.

Initially plenty of food, but as number of microorganisms multiply exponentially it is used up. Nutrient level becomes insufficient to support further growth and reproduction.

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Oxygen levels:
As population rises, so does demand for __________ oxygen, so levels become limiting.

As population rises, so does demand for respiratory oxygen, so levels become limiting.

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Temperature:
______-controlled reactions within microorganisms are affected by temperature of culture medium. Low temp slows down _____ and __________ rate. High temp denatures ______, killing microorganisms.

Enzyme-controlled reactions within microorganisms are affected by temperature of culture medium. Low temp slows down growth and reproduction rate. High temp denatures enzymes, killing microorganisms.

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Build-up of waste:
Bacterial numbers rise, build up of toxic material may inhibit further _____ and can even _____ and kill the culture.

Bacterial numbers rise, build up of toxic material may inhibit further growth and can even poison and kill the culture.

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Change in pH:
as _____ ________ produced by respiration of bacterial cells increases, the pH of the culture falls until a point where ______ activity is affected and population growth is inhibited.

as carbon dioxide produced by respiration of bacterial cells increases, the pH of the culture falls until a point where enzyme activity is affected and population growth is inhibited.

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Both of these methods of operating a bioreactor can be adjusted to ensure the maximum production of ______, or the production of the primary or secondary _______. Most systems are adapted for the maximum yield of ________.

Both of these methods of operating a bioreactor can be adjusted to ensure the maximum production of biomass or the production of the primary or secondary metabolites. Most systems are adapted for the maximum yield of metabolites.

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Methods of immobilising enzymes:
Surface immobilisation - ________ to inorganic carriers, ______ or _____ bonding to inorganic carriers

Surface immobilisation - adsorption to inorganic carriers, covalent or ionic bonding to inorganic carriers

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Entrapment - in the _____, or membrane entrapment in the _________, or behind a semi-permeable membrane

Entrapment - in the matrix, or membrane entrapment in the microcapsules, or behind a semi-permeable membrane

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Using immobilised enzymes:
- glucose _______ can be used to produce fructose from glucose. _______ is much sweeter and is used as a sweetener. Enzyme turns _____ glucose into marketable fructose.

- glucose isomerase can be used to produce fructose from glucose. Fructose is much sweeter and is used as a sweetener. Enzyme turns cheap glucose into marketable fructose.

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- penicillin _____ used to make semi synthetic penicillins. Many bacteria has developed _________ to natural penicillins so are no longer effective drugs. Many bacteria are vulnerable to semi synthetic penicillins, so are important in treating _______ cau

- penicillin acylase used to make semi synthetic penicillins. Many bacteria has developed resistance to natural penicillins so are no longer effective drugs. Many bacteria are vulnerable to semi synthetic penicillins, so are important in treating infectio

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- ______ used to produce lactose-free milk. Some people, and cats are intolerant of lactose. Lactase hydrolyses lactose to ______ and _______, giving lactose-free milk.

- lactase used to produce lactose-free milk. Some people, and cats are intolerant of lactose. Lactase hydrolyses lactose to glucose and galactose, giving lactose-free milk.

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- __________ used to produce pure samples of L-amino acids. Used in the production of pharmaceuticals, organic _______, cosmetics, and ____.

- Aminoacylase used to produce pure samples of L-amino acids. Used in the production of pharmaceuticals, organic chemicals, cosmetics, and food.

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- __________ is used to complete the breakdown of starch to glucose syrup. Amylase breaks starch down into ______. The breakdown of ______ to glucose is catalysed by the enzyme.

- glucoamylase is used to complete the breakdown of starch to glucose syrup. Amylase breaks starch down into dextrins. The breakdown of dextrins to glucose is catalysed by the enzyme.

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Advantages of immobilised enzymes:
- can be _____, which is cheaper
- more reliable, high degree of _____ over the process

- can be reused, which is cheaper
- more reliable, high degree of control over the process

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- greater temperature tolerance, less easily _________ by heat and work at ______ levels over a wider range of temperatures, making the bioreactor less expensive to run.

- greater temperature tolerance, less easily denatued by heat and work at optimum levels over a wider range of temperatures, making the bioreactor less expensive to run.

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Disadvantages of immobilised enzymes:
- reduced efficiency, immobilising enzymes may reduce its ______ rate.
- more _______ than free enzymes, however don't need to be replaced as frequently.

- reduced efficiency, immobilising enzymes may reduce its activity rate.
- more expensive than free enzymes, however don't need to be replaced as frequently.

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- more technical issues, reactors using immobilised enzymes are more complex than simple ________, more things can go wrong.

- more technical issues, reactors using immobilised enzymes are more complex than simple fermenters, more things can go wrong.

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Cloning and Biotechnology M6 C22

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