M6, C22 Cloning and Biotechnology

Question 1

define biotechnology

Answer 1

industrial use of living organisms to produce food, drugs and other products

Question 2

Why are microorganisms used in biotechnology

Answer 2

• ideal growth conditions easily created
• grow rapidly due to short life cycle so products made quickly
• grown on a range of inexpensive materials
• grown anytime of year
• no welfare issues to consider
• large range of microorganisms capable of different chemical synthesis or degradation
• can artificially manipulate them eg. genetic engineering

Question 3

How is lactase used in biotechnology

Answer 3

Breaks down lactose
Made from Asperigillus fungi
Used to produce lactose-free products

Question 4

What are intracellular enzymes

Answer 4

enzymes are contained within cells of a microorganism

Question 5

what are isolated enzymes

Answer 5

enzymes that aren’t contained in cells

• extracellular enzymes which are secreted naturally by the microorganism
• some have to extracted from cells artificially

Question 6

what are the indirect ways microorganisms are used in biotechnology

Answer 6

the actions of the microorganisms are what’s important

eg. bread - yeast makes it rise, yoghurt, cheese, beer

needs ideal conditions
needs to be sterile
the microorganisms used have been genetically engineered
ethical issues

Question 7

what are the direct ways microorganisms are used in biotechnology

Answer 7

Using microorganisms to make proteins we can eat

single-cell protein

eg. Quorn - made of fungus Fusarium venetatum

Question 8

what microorganism is used in baking and how does it work

Answer 8

Yeast (fungi)

The carbon dioxide produced by fermentation of sugars in the dough makes sure it doesn’t stay flat

Question 9

what microorganism is used in brewing and how does it work

Answer 9

Yeast (fungi)
Added to barley (type of grain). The yeast respires anaerobically using the glucose from the grain and produces ethanol and carbon dioxide. (Fermentation)

Question 10

what microorganism is used in making cheese and how does it work

Answer 10

Substance called rennet which contains the enzyme chymosin. This clots milk. It’s extracted from yeast cells that have been genetically modified.

Also, lactic acid bacteria (Lactobacillus or Streptococcus) which converts lactose in milk into lactic acid which contributes to solidifying.

Question 11

what microorganism is used in making yoghurt and how does it work

Answer 11

Lactic acid bacteria
Clots milk and causes it to thicken.
Makes it sour - flavours are added afterwards.

Question 12

What are the advantages of using microorganisms in food production?

Answer 12

• Microorganisms used to make single-cell protein can be grown using many different organic substrates, including waste materials.
• They can be grown quickly, easily and cheaply. Only require simple growth requirements and can be grown on waste products and less land is required in comparison to growing crops or rearing livestock.
• Can be cultured anywhere if you have the right equipment so can be used in LEDCs
• Single-cell protein is also often considered a healthier alternative to animal protein

Question 13

What are the disadvantages of using microorganisms in food production?

Answer 13

• Other microorganisms can grow in the conditions so it takes a lot of effort into making sure food doesn’t get contaminated with unwanted bacteria
• People may not like the idea of eating food that has been grown using waste products
• Single-cell protein doesn’t have the same texture/flavour as real meat
• If single-cell protein is consumed in high quantities, health problems could be caused due to high levels of uric acid released when the large amounts of amino acids are broken down.

Question 14

what are the aims of fermentation vessels

Answer 14

obtain lots of microorganisms or to collect lots of useful product the microorganism makes

Question 15

what is batch fermentation

Answer 15

Microorganisms are grown in individual batches, 1 culture ends, its removed and then a different batch is grown in the vessel
This is known as closed culture

Question 16

what is continuous fermentation

Answer 16

Microorganisms are continually grown in a fermentation vessel without stopping
Nutrients are added and waste products are removed at a constant rate
Conditions inside are kept at the optimum for growth – this maximises yield
This is known as open culture

Question 17

what 5 things must be controlled in fermentation vessels

Answer 17

pH
temperature
oxygen supply
nutrient concentration
contamination

Question 18

why must pH be controlled in fermentation vessels?

Answer 18

if waste products of microorganisms e.g., carbon dioxide build up then pH of mixture will decrease. Change in pH can affect enzyme action and stop growth, buffers are added to mixture and stirred in or alkaline solution added to maintain optimum pH.

Question 19

why must temperature be controlled in fermentation vessels?

Answer 19

if temperature too low microorganisms will not grow quickly enough, too high and enzymes will start to denature and microorganisms are inhibited or destroyed.
A water jacket surrounds the fermentation vessel to maintain the temp.

Question 20

why must oxygen supply be controlled in fermentation vessels?

Answer 20

if microorganisms use up oxygen they will start to die off so need a mechanism to keep nutrient medium oxygenated, oxygen is bubbled through broth when probes or sample tests indicate that levels are dropping to be mixed in using stirrers/paddles as will not spread through fast enough by diffusion alone.

Question 21

why must nutrient concentration be controlled in fermentation vessels?

Answer 21

if microorganisms use up food supply they will start to die off so need a mechanism to keep food supplied, culture medium can be added in controlled amounts to broth when probes or sample tests indicate that levels are decreasing to be mixed in using stirrers/paddles as will not spread through fast enough by diffusion alone.

Question 22

why must contamination be controlled in fermentation vessels?

Answer 22

Vessels are sterilised between uses with superheated steam to kill any unwanted organisms and make sure the next culture isn’t contaminated.
This increases the product yield because the microorganisms aren’t competing with other organisms.

Question 23

Fermentation vessels may run batch or continuous processes. What is the difference? (6)

Answer 23

Continuous processes run continuously once fermentation is started (1);
sterile nutrient medium added continuously once culture is growing exponentially (1);
culture broth continually removed so product can be processed and culture volume remains the same (1).
Batch process everything added at beginning in fixed volume of medium (1);
nutrients used up and microorganisms, products, and waste products build up (1);
may be stationary phase when secondary metabolites formed, process stopped, products extracted, reactor cleaned, and new process begun (1).

Question 24

define closed culture

Answer 24

The growth of microorganisms in an environment where the conditions are fixed. No new nutrients are added and waste products are not removed.

Question 25

In a closed culture, a population of microorganisms follows a standard growth curve. What are the 4 phases and describe what’s happening.

Answer 25

1) The lag phase - where there is no reproduction but the cells are taking up water and are carrying out protein synthesis and producing enzymes.
2) The exponential phase or log phase - where the population increases rapidly.
3) The stationary phase - where bacterial cells are dying at the same rate as they are produced. Nutrients decrease, waste builds up.
4) The death or decline phase - where more cells are dying than are being produced. Nutrients are exhausted, toxic levels of waste.

Question 26

What would the growth curve look like for continuous fermentation? How does it differ to the standard growth curve?

Answer 26

Horizontal line (lag phase) then straight line upwards (exponential/log phase).

Missing the stationary phase and death phase because the waste products are removed and oxygen and nutrients are continuously supplied.

Question 27

What equation could you use to work out how many cells will be present in a population after a certain number of divisions?

Answer 27

No. of cells in population = initial no. of cells x 2^n

n is the number of divisions

Question 28

For a species of bacteria, the cells divide approximately every 30 minutes during the exponential growth phase. There are 300 bacterial cells present at the start of the phase. Estimate how many cells are present after 7 hours.

Answer 28

7 hours = 420 minutes
Divide every 30 minutes so 420/30 = 14 divisions

No. of cells in population = initial no. of cells x 2^n
= 300 X 2^14
= 4915200
= 4.9 X10^6 cells

Question 29

why is it easier to interpret data values from a logarithmic graph when looking at growth rate of microorganisms

Answer 29

because on a normal linear graph, the numbers are very clumped together at the start as the y axis has to cover a wide range of values

Question 30

If you had a log graph showing the growth rate of a microorganism, how could you read how many microorganisms are present at a certain time?

Answer 30

Read the time from x axis and go up and across to the y axis

the number you read will be the log value so you have to do 10^x to calculate the actual number of microorganisms

Question 31

How is penicillin produced

Answer 31

Mould called Penicillin chrysogenum.
Needs relatively high oxygen levels and a rich nutrient medium.
Semi-continuous batch process is used. Fungus grows in 1st stage and penicillin grows in 2nd stage.

Uses small fermenters to maintain high levels of oxygenation.
Mixture is continuously stirred to keep it oxygenated.
Rich nutrient medium.
Contains a buffer to maintain pH.
Bioreactors maintain temperature.

Question 32

How is insulin produced as part of biotechnology

Answer 32

Insulin is made by genetically modified bacteria which have had the gene for human insulin production inserted into their DNA.
These bacteria are grown in an industrial fermenter on a massive scale and the insulin produced is collected and purified.

Question 33

What is bioremediation

Answer 33

Using microorganisms to remove pollutants, like oil and pesticides, from contaminated sites.

Can use naturally occurring microorganisms which could provide nutrients to break down/neutralise contaminants.
Or can use GM organisms which break down contaminants which they woudlnt naturally encounter.

Question 34

Give examples of all the aseptic techniques that should be carried out when culturing microorganisms

Answer 34

• Regularly disinfect work surfaces to minimise contamination.
• Work near a Bunsen flame so the hot air removes the microorganisms in the air.
• Sterilise the instrument used to transfer cultures before and after each use. Eg. put the inoculation loop through a Bunsen flame.
• If you’re using broth, briefly pass the neck of the broth container through a flame just after it’s opened and just before it’s closed.
• Minimise the time that the agar plate is open and put the lid on as soon as possible.
• Sterilise all glassware before and after use.
• Wear a lab coat and gloves. Tie long hair back.

Question 35

Outline an experiment you can do to culture microorganisms.

Answer 35

1) Bacteria should be in a broth. Using a sterile pipette, add the same volume of your sample to each of the 6 agar plates. Discard pipette after use.
2) Spread the broth across the entire surface of the agar using a sterile plastic spreader.
3) Put the lids on the agar plates and tape them shut.
4) Place 3 plates in a fridge at 4 degrees and put 3 in an incubator at 25 degrees. Should be incubated upside down to stop condensation forming and dropping onto the agar.
5) Put another lidded agar plate in each of the 2 different temperatures which are uncultured. These are negative controls.
6) Leave all the plates for the same amount of time (24hours)
7) Observe the results - there will be colonies of bacteria
8) Count the number of colonies that have formed on each plate and record your results.
9) Work out the mean number of colonies formed at each temp.

Question 36

How could you alter the experiment for culturing microorganisms to investigate the affect of pH

Answer 36

Add buffers at different pH levels to the broth

Question 37

how do you make a serial dilution

Answer 37

1) In your 1st test tube put 2ml of pure culture (no broth)
2) Squeeze with pipette to mix
3) In the remaining 4 test tubes place 9ml of broth
4) From the original inoculum (pure culture), use a clean tip and take 1ml and add to 2nd test tube
5) Squeeze with pipette to mix, take a new tip and take 1ml and add to next test tube
6) Repeat for all 4 dilutions

Question 38

give the procedure of how you sterilise the glass spreader used for spreading the culture on the agar plate

Answer 38

Dip the spreader in the ethanol
Tap twice to remove excess ethanol
Pull the glass spreader pointing downwards slowly through the flame
Do not put this down anywhere
Let the spreader cool down before putting in culture
Ensure you flame the culture bottle before and after use

Question 39

what are the dilution factors of 4 test tubes if the first one has 9ml of broth and 1ml of pure culture and then 1ml of that solution is put in the next 9ml broth test tube and so on.

Answer 39

10^1 then 10^2, 10^3 and 10^4

because test tube one has the ratio 1:10 of culture:broth
test tube 2 is 1:100
test tube 3 is 1:1000
test tube 4 is 1:10000

Question 40

how do you calculate the number of colonies in a sample

Answer 40

Count the number of colonies on the plate you have chosen from each set and record this number and the dilution factor for that plate.

(number of colonies counted x dilution factor) / vol on culture plate

volume is in ml

Question 41

how could you use a colorimeter to find out the density of bacteria in a culture
why isn’t this as good as serial dilutions

Answer 41

Turbidity of the culture can be used, once a calibration curve has been produced from a known set of cultures. The % transmission of light through the culture can be measured with a colorimeter and the density of the unknown culture inferred from the calibration curve. This method takes no account of viability of cells so will give an overestimate, especially in old, high density cultures.

Question 42

When observing your agar plates, how can you be confident that all the colonies you see are the same?

Answer 42

The quickest check to be made is simply colour and morphology of colonies. All colonies should look the same. Any different colonies are almost certainly the result of contamination.
Microscopic observation of samples from any suspect colonies, using identification aids such as Gram-staining, could confirm this.

Question 43

define metabolites

what are the two types

Answer 43

the products of metabolism

primary and secondary

Question 44

what are primary metabolites

Answer 44

Substances made by an organism as part of normal growth

Amino acids, proteins, enzymes, nucleic acids, ethanol and lactate.

These are made during the growth / log phase.

Question 45

what are secondary metabolites

Answer 45

Substances made by an organism that are not part of normal growth.

Antibiotics, such as penicillin

These are produced after the growth phase and are made during the stationary phase.

Question 46

All microbes produce ______ metabolites, but only a select few produce ______ metabolites.

Answer 46

All microbes produce PRIMARY metabolites, but only a select few produce SECONDARY metabolites.

Question 47

Compare batch and continuous cultures

Answer 47

Batch are closed cultures whereas continuous are open cultures.
Phases involved in batch are lag, log, stationary and death phase. But, phases in continuous are just log and lag.
Primary and secondary metabolites are produced in batch but only primary metabolites are produced in continuous.

Question 48

why do you get primary and secondary metabolites in batch fermentation

Answer 48

During the growth phase, there are plenty of nutrients and therefore all metabolic processes are dedicated to growth and so primary metabolites are made that aid the growth of the organism, such as proteins.

In the stationary phase, when nutrients are limiting, microbes switch to produce secondary metabolites, such as antibiotics. These will help to kill other microbes which could be in competition with the it.

Question 49

simply, what is immobilisation of enzymes

Answer 49

any technique where enzyme molecules are held and separated from the reaction mixture

Substrate molecules can bind to the enzyme molecules and the products formed go back into the reaction mixture leaving the enzyme molecules in place.

Question 50

what are the 4 ways you can immobilise enzymes

Answer 50

1) Encapsulated/entrapped in jelly-like alginate beads, which act as a semi-permeable membrane.
2) Trapped in a silica gel matrix.
3) Covalently bonded to insoluble material like cellulose or collagen fibres.
4) Adsorption - enzyme molecules adsorbed to inorganic carriers e.g. cellulose, silica

Question 51

how do columns of immobilised enzymes work

Answer 51

The substrate solution is run through a column of immobilised enzymes

The active sites of the enzymes are still able to catalyse the reaction

The solution flowing out of the column will only contain the desired product

Question 52

what are the advantages of using immobilised enzymes

Answer 52

• columns of immoblised enzymes can be washed and reused - this reduces the cost of running a reaction on an industrial scale because you don’t have to keep buying new enzymes
• the product isn’t mixed with the enzymes - no money or time is spent separating them out.
• immobilised enzymes are more stable than free enyzmes - they’re less likely to denature (become inactive) in high temperatures or pH extremes

Question 53

what are the disadvantages of using immobilised enzymes

Answer 53

• extra equipment is required which can be expensive to buy
• immobilised enzymes are more expensive to by than free enzymes, so coupled with the equipment costs, they’re not always economical for use in smaller-scale production
• the immobilisation of the enzymes can sometimes lead to a reduction in the enzyme activity because they can’t freely mix with their substrate

Question 54

how are immobilised enzymes used to convert lactose to glucose and galactose

Answer 54

Eg. milk

Fresh milk can be passed over immobilised lactase to produce lactose-free milk.
Lactase breaks down lactose into glucose and galactose via a hydrolysis reaction.

Question 55

how are immobilised enzymes used to produce semi-synthetic penicillins

Answer 55

some bacteria are resistant to penicillin so semi-synthetic penicillin has been produced which has an effect

immobilised penicillin acylase enzyme is used

Question 56

how are immobilised enzymes used to convert dextrins to glucose

Answer 56

glucose can be derived from starchy food with the help of immobilised enzymes
starch breaks down into dextrins which are then broken down into glucose by the immobilised enzyme glucoamylase

Question 57

how are immobilised enzymes used to convert glucose to fructose

Answer 57

fructose can be used instead of glucose - you need less for the same level of sweetness

immobilised glucose isomerase is used

Question 58

how are immobilised enzymes used to produce pure samples of L-amino acids

Answer 58

amino acids have 2 chemical forms - L or D

most amino acids utilised by the body need to be in the L form

scientists are able to chemically synthesise amino acids, but end up with a mix of L and D forms

the enzyme aminoacylase separates them

L-amino acids can then be used for lots of things like dietary supplements or animal/human food

Question 59

why are immobilised enzymes used to convert acrylonitrile to acrylamide

Answer 59

acrylamide is a chemical that is typically used in industry to produce synthetic polymers

immobilised nitrilase is used to covert acrylonitrile (a man-made chemical) to acrylamide