Unit 2 (KA6-7)

Question 1

Prokaryote

Answer 1

Organism lacking a true nucleus, eg. bacteria, archaea

Question 2

Eukaryote

Answer 2

Cells containing a true nucleus, eg. plant, animal and fungal cells

Question 3

Properties of micro-organisms

Answer 3

They are highly adaptable and can grow on a variety of substrates, including waste materials.

They make a wide range of metabolic products which are useful to humans. eg. alcohol, antibiotics.

They are easy to cultivate - they grow and reproduce rapidly.

Question 4

Growth medium

Answer 4

A substance used for culturing micro-organisms.

Can be solid agar or liquid broth.

Various substances can be added to control the metabolism of the micro-organisms being grown.

Question 5

Composition of growth media

Answer 5

Carbohydrates (eg. starch) - needed as an energy source and to build all organic molecules.

Water - source of hydrogen and oxygen for building molecules. Oxygen is required for respiration.

Nitrates and phosphates - needed to build amino acids, DNA and ATP.

Sulfates - needed for building some amino acids

Beef extract - contains vitamins and fatty acids which cannot be synthesised by some micro-organisms.

Enzyme inhibitors and inducers may also be added to divert metabolic pathways.

Question 6

Biosynthesis

Answer 6

The production of complex molecules within cells.

Question 7

Aseptic technique

Answer 7

Sterile conditions used when preparing and inoculating growth media with micro-organisms.

Includes using disinfectant, heating equipment in a flame and protective clothing.

Prevents contamination with rival microbes which may cause spoilage of the product.

Question 8

Fermenter

Answer 8

A container holding a large volume of liquid growth medium in monitored and controlled conditions.

Temperature, oxygen supply and pH are carefully controlled.

Used to grow microbes in optimum conditions on a large scale to produce antibiotics, enzymes etc.

Question 9

Sensors/probes

Answer 9

Used to monitor conditions inside a fermenter, so that optimum conditions can be maintained.

Information is fed back to a computer which can alter factors such as the flow rate of cooling water, to adjust the temperature inside the fermenter.

Question 10

Paddle/stirrer

Answer 10

Blades inside a fermenter that are driven by a motor.

Stirs the culture to prevent the cells from sinking or settling.

Question 11

Growth

Answer 11

The rate of production of new cells is faster than the rate of cell breakdown, resulting in an increase in biomass (living tissue).

Question 12

Measuring microbial growth

Answer 12

The cells in samples taken from the culture are dried and weighed to measure biomass.

Alternatively, cell numbers in a known volume can be counted using a haemocytometer.

Question 13

Mean generation time (doubling time)

Answer 13

The time taken for a population of micro-organisms to double in number.

Question 14

Semi-logarithmic graph paper

Answer 14

Has a normal linear scale along the x-axis and a logarithmic scale along the y-axis. (1-10, 10-100, 100-1000 etc)

This allows a large range of values to be plotted.

Microbial populations increase rapidly and are difficult to plot on a normal graph.

Question 15

Phases of growth

Answer 15

The pattern shown by microbial populations in culture, when number of viable cells is plotted on semi-log graph paper against time.

Shows population growth in 4 phases - lag, log, stationary, death

Question 16

Lag phase

Answer 16

Small or no increase in cell number - the graph remains low and level.

The cells adjust to the growth medium, and their metabolic rate increases as they induce enzymes to prepare for growth.

Question 17

Log phase

Answer 17

A period of exponential growth at the maximum rate possible.

The number of cells doubles with each division, so the population increases rapidly and the graph goes steeply upwards.

Nutrients are plentiful and there are no other limiting factors.

Question 18

Stationary phase

Answer 18

Rate of new cell production = rate of cell death, and the graph levels out.

Nutrients begin to run out, toxic waste products and secondary metabolites start to build up, and the rate of cell division decreases.

Question 19

Death phase

Answer 19

More cells are dying than are being produced, due to lack of nutrients and accumulation of toxic products.

Can only be seen if viable cells are counted (as dead ones remain in the culture).

The number of viable cells drops and can be seen on the graph.

Question 20

Viable cell count

Answer 20

Only living cells are counted.

Allows a death phase to be seen.

Question 21

Total cell count

Answer 21

All cells, viable and dead, are counted.

The death phase cannot be seen in a total cell count.

Question 22

Haemocytometer

Answer 22

A specialised microscope slide used to count the number of cells in a sample of known volume.

Question 23

Primary metabolism

Answer 23

Occurs during periods of active growth (lag and log phases)

Substrates are broken down to release energy and produce molecules such as amino acids which are essential for growth.

Question 24

Secondary metabolism

Answer 24

Produces substances that are not used for growth, but which may give an ecological advantage. eg. antibiotics

Happens during stationary phase, when the culture is under pressure and nutrients are running out.

Used to kill rival microbes which may compete for limited resources.

Many secondary metabolites are very useful to humans eg. penicillin (antibiotic) and cyclosporin (immunosuppresant drug)

Question 25

Mutagenesis

Answer 25

The creation of mutations by deliberately exposing an organism to mutagenic agents such as UV light, other types of radiation or chemicals, which alter their DNA.

Question 26

Wildtype

Answer 26

The original strain of the micro-organism.

Mutants can revert to wildtype by repairing mutations in their DNA, so improved strains used in industry need constant monitoring.

Question 27

Mutant strain

Answer 27

An improved strain of a micro-organism created by mutagenesis.

It may lack a key enzyme, which may stop or divert a metabolic pathway, leading to accumulation of a desired product.

Mutant strains tend to be unstable and can revert to wildtype.

Question 28

Recombinant DNA technology

Answer 28

Genetic engineering.

A selected gene can be transferred into a host organism using a vector

The host organism is then able to copy and express the new gene, producing the desired protein.

Question 29

Recombinant DNA

Answer 29

Host DNA and donor DNA combined together.

eg. a bacterial plasmid with a human gene inserted into it.

Question 30

Restriction endonuclease.

Answer 30

DNA cutting enzymes, used to cut genes out of donor DNA and used to cut plasmids open.

There are many different types eg. EcoR1 (named after E.coli where it is found)

Each type recognises and cuts at a different specific DNA sequence.

Cuts may be straight or staggered, creating ‘sticky ends’

Question 31

DNA ligase

Answer 31

An enzyme which is used to join fragments of DNA together. eg. it seals the donor gene into the plasmid.

Question 32

Vector

Answer 32

A bacterial plasmid or artificial chromosome.

Used to carry the donor gene into the host organism.

Question 33

Artificial chromosomes.

Answer 33

Constructed by scientists.

Have the same features as a plasmid vector but can carry much longer DNA sequences into the host organism.

Question 34

Features of vectors

Answer 34

Origin of replication

Marker gene

A restriction site

Question 35

Restriction site

Answer 35

Contains target (recognition) sequences for specific restriction endonucleases.

The same restriction endonuclease must be used to cut the donor DNA and the plasmid, so that the sticky ends are complementary and the 2 pieces of DNA can be joined together.

Question 36

Marker gene

Answer 36

Allows scientists to select only the cells that have taken up the recombinant plasmids, to avoid wasting resources.

Ampicillin (antibiotic) resistance is an example of a marker gene.

It is included in the vector so that when the culture is treated with an antibiotic, only the cells containing the plasmid survive, as they have obtained the resistance gene along with the required donor gene.

Cells which don’t have the plasmid are destroyed.

Question 37

Origin of replication

Answer 37

Essential for the replication of the recombinant plasmid in the transformed host cell.

Contains genes that control the self replication of the plasmid or artificial chromosome.

Also contains regulatory sequences that control expression of existing and inserted genes.

Question 38

Limitations of prokaryotes

Answer 38

Prokaryote DNA does not contain introns, so bacterial cells cannot splice eukaryotic DNA (which does contain introns).

Eukaryotic polypeptides (such as insulin) may not be cut or folded correctly after they have been translated, as prokaryotes lack the machinery to do this.

Question 39

Recombinant yeast

Answer 39

Yeast contains plasmids which can be modified using recombinant DNA technology.

Yeast is harder to grow than bacteria, but it is eukaryotic and so is able to carry out RNA splicing and modification of polypeptides.

Question 40

Handling micro-organisms

Answer 40

Ethical considerations and safety need to be taken into account when altering micro-organisms.

Additional genes may be added to the plasmid as a safety mechanism to prevent the GM organism surviving outside the lab.

Aseptic technique should always be used, and additional containment steps may be needed to prevent escapes.