APS138 Cell And Molecular Biology - Smith Flashcards
What is biotechnology?
The manipulation of organisms or their components to make useful products - for our benefit
How is DNA isolated in microbes?
Heat/alkaline lysis
How is DNA isolated in higher organisms?
Mechanical disruption of cells and extraction with salt, a buffer and detergent
When was amplification using PCR invented?
1983
What are the 3 stages of PCR?
Denaturation (95 degrees)
Annealing (variable temp)
Extension (72 degrees and taq DNA polymerase) - 5’ to 3’ direction
What goes into the PCR reaction (5 things)?
Template DNA Primers dNTPs Buffer Taq DNA polymerase
What is DNA ligation used for?
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
What are restriction enzymes?
Enzymes used by bacteria as a defence mechanism against foreign DNA
Over 600 commercially available
4 main types
What do commercially available restriction enzymes usually cut?
Palindromic sequences 4-8bp long
Why are cloning vectors (plasmids) required?
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
What is transformation?
Putting the gene of interest into a bacterium
How is transformation carried out?
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)
What is selection?
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
What does ampicillin inhibit?
Transpeptidase, cell wall synthesis
Why do we use bacterial screening?
To check that the gene of interest is in the cloning vectors in the bacteria
Describe blue/white bacterial screening.
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.
There are two types of selection/screening involved. What are they?
Antibiotic selection for the presence of the cloning vector
Blue/white screening for absence/presence of an insert in the cloning vector.
What are the 5 basic stages of biotechnology?
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)
What can biotechnology do?
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
What was the first protein to be sequenced?
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
What are the two ways insulin can be made?
Expression in E.coli or yeast
How is insulin produced in bacteria?
Chains A and B are produced from two plasmids in two bacterial strains, the chains are purified and then linked with disulfide bonds
How is insulin made from yeast?
Proinsulin is produced, then disulfide bonds are formed and a proteases removes the 33 amino acid chain C + purification
What does cheese production require?
Proteases - traditionally isolated as a complex of enzymes from veal calf stomachs (rennet)
What gene is transferred to bacteria and fungi so that enzymes for cheese production can be translated?
Chymosin B
What are the advantages for using biotechnology instead of veal stomachs for cheese production?
Higher yield, better texture, less bitter, social considerations (vegetarian, halal, kosher, but not organic)
What are the advantages of plant biotechnology?
Increased productivity, increased storage of crop, increased nutritional or taste attributes, disease resistance, nitrogen fixation for crops, stress resistance
When is salicylic acid mostly activated in plants?
When attacked by biotrophic pathogens (which parasitise living plant cells
What does the perception of MAMPs (or PAMPs) trigger?
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
What do necrotrophic pathogens do?
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
What does SA work as in animals? Plants?
Immuno-suppressant in animals, immune stimulant in plants
Which is the constitutive expressor gene of GUS?
p35S:GUS
Which is the SA-inducible GUS gene?
pPR1:GUS
What is the fungus used in the practical?
Plectosphaerella cucumerina
Why do the leaves produce bubbles under a vacuum?
Air removed from airspaces between the mesophyll cells in the leaves
What does SA cause?
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
