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
Antibiotic selection:
Ampicillin (if using pGEM-T) selects for bacteria that contain the PLASMID. If the plasmid is present then the bacterium can multiply to form a colony.
In antibiotic selection: the antibiotics used will depend on the
selection gene:
- Ampicillin (Amp^R)
- Kanamycin (Kan^R)
- Spectinomycin (Spec^R)
in antibiotic selection:
no plasmid =
no growth on antibiotic
plasmid = growth on antibiotic
How do antibiotics work?
Ampicillin
mechanism: Inhibits transpeptidase, cell wall synthesis.
Resistance: Beta-lactamase hydrolyses cleavage of the Beta -lactam ring
How do antibiotics work?
Kanamycin
Mechanism: Inhibits 30S ribosomal subunit, protein synthesis
Resistance: Inactivates antibiotic through phosphorylation
How do antibiotics work?
Spectinomycin
Mechanism: Inhibits 30S ribosomal subunit translocation, protein synthesis
Resistance: Inactivates antibiotic through adenylylation
How do antibiotics work?
Rifampicin
Mechanism: Inhibits bacterial DNA-dependant RNA polymerase
Resistance: Mutations in the polymerase
We have selected for bacterial colonies containing the plasmid…
but does the plasmid have our gene of interest ligated into it?
Bacterial screening is
checking the plasmid has our gene of interest in it
Selection is combined with
Blue/white screening
Insertion site in the plasmid is in the
LacZ gene
LacZ gene = Beta-galactosidase
this gene encodes an enzyme involved in the breakdown of lactose
The transformed E. Coli strain (e.g. DH5alpha) has a mutation in its own
LacZ gene (LacZ(triangle)M15) -the lacZ gene encodes full-length Beta-galactosidase. The lacZ fragment of LacZ(triangle)M15 genes both code for parts of LacZ which can interact to form active protein
IPTG (Isopropyl Beta-D-1-thiogalactopyranoside) is a
non-metabolised lactose analogue that induces lacZ expression.
Bacterial screening: bacterial chromosome with LacI and LacZ(triangle)M15
LacI produces inactive repressor
LacZ(triangle)M15 produces inactive Beta-galactosidase
Plamid LacZ fragment joins with inactive Beta-galactosidase =
Active beta-galactosidase
Blue/White screening:
Alternative substrate of X-gal is provided to the bacteria.
X-gal (colourless) –Beta-galactosidase–> D-galactosidase + other –> Blue product
Insert in lacZ gene =
no lacZ = WHITE COLONIES
No insert =
lacZ produced = BLUE COLONIES
On Amp + IPTG +X-gal with NO PLASMID
- no plasmid
- no resistance to Amp
- No colony
On Amp + IPTG +X-gal with no insert in the plasmid
- no insert
- active Beta-galactosidase
- BLUE colony
On Amp + IPTG +X-gal with insert in the plasmid
- insert
- Inactive Beta-galactosidase
- WHITE colony
__ types of selection/screening
TWO
____ selection for RRESENCE of the ____
ANTIBIOTIC
PLASMID
___ for ABSENCE/PRESENCE of an __ in the PLASMID
BLUE / WHITE SCREENING
INSERT
