APS138 Cell And Molecular Biology - Smith

Question 1

What is biotechnology?

Answer 1

The manipulation of organisms or their components to make useful products - for our benefit

Question 2

How is DNA isolated in microbes?

Answer 2

Heat/alkaline lysis

Question 3

How is DNA isolated in higher organisms?

Answer 3

Mechanical disruption of cells and extraction with salt, a buffer and detergent

Question 4

When was amplification using PCR invented?

Answer 4

1983

Question 5

What are the 3 stages of PCR?

Answer 5

Denaturation (95 degrees)
Annealing (variable temp)
Extension (72 degrees and taq DNA polymerase) - 5’ to 3’ direction

Question 6

What goes into the PCR reaction (5 things)?

Answer 6

Template DNA
Primers
dNTPs
Buffer
Taq DNA polymerase

Question 7

What is DNA ligation used for?

Answer 7

To join pieces of DNA - an enzyme called DNA ligase is used to catalyse phosphodiester bond formation between nucleotides - much more efficient with sticky ends than blunt ends

Question 8

What are restriction enzymes?

Answer 8

Enzymes used by bacteria as a defence mechanism against foreign DNA
Over 600 commercially available
4 main types

Question 9

What do commercially available restriction enzymes usually cut?

Answer 9

Palindromic sequences 4-8bp long

Question 10

Why are cloning vectors (plasmids) required?

Answer 10

Cloned genes need additional DNA and genes to be replicated and expressed in the bacteria (if desired).
e.g. antibiotic resistance for selection later on in the process

Question 11

What is transformation?

Answer 11

Putting the gene of interest into a bacterium

Question 12

How is transformation carried out?

Answer 12

Gene of interest ligated into cloning vector (plasmid). Ligation mixture added to competent cells (usually E. coli). Cells heat shocked at 42 degrees for 1 min then allowed to recover at 37 in a rich medium. After recovery cells spread onto a solid media and allowed to grow with selection.

Alternatively competent cells can be transformed via an electric current (electroporation rather than heat shock)

Question 13

What is selection?

Answer 13

Selecting bacteria that contain the plasmid (cloning vector) - if present bacteria can multiply to form a colony.
Antibiotics such as ampicillin, kanamycin or spectinomycin could be used - if resistant genes inserted into cloning vector earlier on

Question 14

What does ampicillin inhibit?

Answer 14

Transpeptidase, cell wall synthesis

Question 15

Why do we use bacterial screening?

Answer 15

To check that the gene of interest is in the cloning vectors in the bacteria

Question 16

Describe blue/white bacterial screening.

Answer 16

Insertion site in the plasmid is in the lacZ gene (codes for B-galactosidase) - encodes enzyme involved in the breakdown of lactose. Transformed E. coli strain has mutation in its lacZ gene - lacZ fragment and mutated fragment both code for parts of lacZ which can interact to form an active protein. Alternative sunstrate of X-gal is provided to the bacteria (colourless) - insert in lacZ gene = no lacZ = white colonies. No insert = lacZ produced = Blue colonies.

Question 17

There are two types of selection/screening involved. What are they?

Answer 17

Antibiotic selection for the presence of the cloning vector

Blue/white screening for absence/presence of an insert in the cloning vector.

Question 18

What are the 5 basic stages of biotechnology?

Answer 18

Obtain genome. Amplify gene of interest (PCR). Get gene into useful form using restriction enzymes, ligases and cloning vectors. Insert gene into bacterium (transformation). Making sure this process was successful (selection & screening)

Question 19

What can biotechnology do?

Answer 19

Improve the human condition through:

• treatment of disease
• industrialisation/resource efficiency, e.g. of food processes, clothing and washing industry
• caring for our environment
• Improving nutrition

Question 20

What was the first protein to be sequenced?

Answer 20

Insulin - normally produced in pancreatic B-cells to remove excess glucose from the blood - injections control type I (usually) diabetes - used to be isolated from bovine and swine pancreas

Question 21

What are the two ways insulin can be made?

Answer 21

Expression in E.coli or yeast

Question 22

How is insulin produced in bacteria?

Answer 22

Chains A and B are produced from two plasmids in two bacterial strains, the chains are purified and then linked with disulfide bonds

Question 23

How is insulin made from yeast?

Answer 23

Proinsulin is produced, then disulfide bonds are formed and a proteases removes the 33 amino acid chain C + purification

Question 24

What does cheese production require?

Answer 24

Proteases - traditionally isolated as a complex of enzymes from veal calf stomachs (rennet)

Question 25

What gene is transferred to bacteria and fungi so that enzymes for cheese production can be translated?

Answer 25

Chymosin B

Question 26

What are the advantages for using biotechnology instead of veal stomachs for cheese production?

Answer 26

Higher yield, better texture, less bitter, social considerations (vegetarian, halal, kosher, but not organic)

Question 27

What are the advantages of plant biotechnology?

Answer 27

Increased productivity, increased storage of crop, increased nutritional or taste attributes, disease resistance, nitrogen fixation for crops, stress resistance

Question 28

When is salicylic acid mostly activated in plants?

Answer 28

When attacked by biotrophic pathogens (which parasitise living plant cells

Question 29

What does the perception of MAMPs (or PAMPs) trigger?

Answer 29

A signalling cascade, which activates transcription factors that migrate to the nucleus, where they bind to gene promoters of genes involved in the biosynthesis of SA - accumulates in the cytoplasm

Question 30

What do necrotrophic pathogens do?

Answer 30

Kill plant cells and use the dead tissue as substrate to colonise the plant - they are insensitive to SA-dependent defences.
Switch from biotrophic lifestyle to necrotrophic after a couple of days to escape SA-dependent immune responses

Question 31

What does SA work as in animals? Plants?

Answer 31

Immuno-suppressant in animals, immune stimulant in plants

Question 32

Which is the constitutive expressor gene of GUS?

Answer 32

p35S:GUS

Question 33

Which is the SA-inducible GUS gene?

Answer 33

pPR1:GUS

Question 34

What is the fungus used in the practical?

Answer 34

Plectosphaerella cucumerina

Question 35

Why do the leaves produce bubbles under a vacuum?

Answer 35

Air removed from airspaces between the mesophyll cells in the leaves

Question 36

What does SA cause?

Answer 36

Rapid fluctuations in the redox state within the cytoplasm - partially reduces complexes of defence regulatory protein NPR1 into monomers that move into the nucleus and recruit transcription factors that initiate defence gene transcription