APS138 Cell And Molecular Biology - Smith Flashcards
(36 cards)
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)
