Lecture 9: Biotechnology 1 Flashcards
Biotechnology is
- The manipulation of organisms or their components to make useful products
- The manipulation organisms and their components for our benefit
- to make use of biological resources, we need tool and processes
Basic starting material for biotechnology process’
Genomes
microbial genomes are ___ bp
1 X 10^5 to 1 X 10^7 bp
Human genome is __ bp
3 X 10^9 bp
DNA isolation for microbes:
heat or alkaline lysis
DNA isolation for higher organisms than microbes:
- mechanical disruption of cells
- extraction of DNA with salt, buffer and detergent
more sophisticated methods of DNA isolation:
- binding of DNA to silica membranes
- removal or proteins with phenol:chloroform
- DNA purification by CsCl gradient
Process used to amplify your genes:
Polymer Chain reaction - PCR
PCR first invented in
1985
PCR steps:
1) Select sequence of interest
2) Denaturation (splitting of strands) at 94-98 degrees Celsius
3) Annealing of primers (variable degrees) (primers around 20nt)
4) Extension 72 degrees - Taq DNA polymeras 5’–>3’
what goes into PCR reaction:
- template DNA
- Primers
- dNTPs
- Buffer
- Taq DNA polymerase
to join pieces of DNA an enzyme called
DNA LIGASE is used
what does DNA ligase do
catalyses phosphodiester bond formation between nucleotides
reaction involving DNA and DNA ligase
separate DNA –DNA ligase–> linked DNA.
ATP is converted to AMP + PPi
DNA ligase is much more efficient at joining
sticky ends than blunt ends
Restriction enzymes are used by bacteria
as a defence mechanism against foreign DNAs
Isolated for using in DNA cloning over __ commercially available
600
how many types of restriction enzymes are there?
4
examples:
Palindromic, (dis-)continuous, 4-8bp & Palindromic & dicsontinous most commonly used
commercially available restriction enzyme usually cut :
palindromic sequences 4-8 bp long
Cloning vectors provide
the additional DNA and genes needed for the cloned genes to be replicated & expressed (if desired) in the bacteria
Cloning vector example:
-plasmid name & total size of plasmid in centre
-Amp^r = gene to select for transformed cells - codes for resistance to the antibiotic Ampicillin
Second origin of replication allows production of ssDNA
-LacZ gene = beta-galactosidase
-gap where you insert your gene or DNA of interest. Taq DNA polymerase adds an extra Adenine (A) to each end which can be used for cloning.
-Ori = origin of replication - allows plasmid to be replicated up to 300-350 copies per cell
Transformation of bacteria: 1st way
- Genes of interest ligated into the cloning vector. Need to put it into bacterial cells for amplification.
- Ligation mixture added to competent cells (usually non-pathogenic E. coli)
- Cells “heat-shocked” at 42 degrees for 1 minute, then allowed to recover at 37 degrees in a rich medium (Luria Broth = LB)
- After recovery, cells are spread on to solid media and allowed to grow with selection
Transformation of bacteria: 2nd way
- Alternatively competent cells can be transformed via an electric current
- This is referred to as “electroporation” rather than “heat shock”
- Cells still require recovery in a rich medium before being spread on to the solid media plate and selected
to make sure you really did get your gene of interest into the bacterium (selection) you must run selection and screening in
parallel
