Recombinant DNA technology II Flashcards
(Transformation) how do we make cells competent
Modified DNA is inserted in bacteria via transformation
(transfection for eukaryotic cells)
- Chemical transformation
-chill cells in CaCl2 to permeabilise membrane
-heat shock to prompt DNA uptake - Electroporation
-purify cells to remove ions
-high voltage shock makes holes in membrane - After uptake of DNA, cells need time to recover before selection
Bacteria culture
- Culture bacteria on agar plate
- Spread culture single cells enough to obtain single colonies
(clones) - From each single colony make liquid cultures for subsequent use/analyses.
NB to sterile techniques
each dot on the agar is a genetically identical colony so that’s why we spread the solution at the start to make sure each bacterium is far from others.
Selection of bacteria with insert
(don’t need to memorise)
Select insert-containing colonies via
* antibiotics, e.g.
* penicillins (ampicillin), target cell wall
* chloramphenicol, targets protein synthesis
* tetracycline, targets protein synthesis
* aminoglycosides (streptomycin), target protein synthesis
* ansamycin (rifamycin), targets transcription
* white/blue colonies
Inhibitors of transcription/translation
(don’t need to memorise)
*Acting only on bacteria
Tetracycline blocks binding of aminoacyl-tRNA to A-site of ribosome
Streptomycin prevents the transition from initiation complex to chain-elongating ribosome and also
causes miscoding
Chloramphenicol blocks the peptidyl transferase reaction on ribosomes
Erythromycin blocks the translocation reaction on ribosomes (step 3 in Figure 6-65)
Rifamycin blocks initiation of RNA chains by binding to RNA polymerase (prevents RNA synthesis)
*Acting on bacteria and eukaryotes
Puromycin causes the premature release of nascent polypeptide chains by its addition to growing chain end
Actinomycin D binds to DNA and blocks the movement of RNA polymerase (prevents RNA synthesis)
*Acting only on eukaryotes
Cycloheximide blocks the translocation reaction on ribosomes
Anisomycin blocks the peptidyl transferase reaction on ribosomes
α-Amanitin blocks mRNA synthesis by binding preferentially to RNA polymerase II
Select with white/blue colonies (lac operon)
Plasmid with lac-Z:
* Functional β-galactosidase produced by bacteria
* lac → glc + gal
β-galactosidase breaks down lactose in glucose and galactose it also breaks down x-gal
*Chromogenic X-Gal (something that can be coloured)(5-bromo-4-chloro-indolyl-β, added to agar plate) → 5-bromo-4-chloroindigo → bright blue colony
* if lacZα sequence in MCS (plasmid with insert), β-gal is disrupted → no functional β-galactosidase → white colony
Blue colonies:
with functional β-gal → no insert
White colonies: no functional β-gal → with insert
Screening: pick white colonies
(non-functional β-gal and => the insert)
How to screen for the right insert?
You have all conies with insert but how do you know if it is correct e.g correct orientation
Screen for correct insert
You have selected the colonies which contain an insert (e.g.
white colonies), but which ones contain the correct insert, in
the correct orientation?
two methods
* restriction digest with correct REs, run on agarose gel
electrophoresis, check insert size
* colony-PCR: PCR amplify the insert, run on agarose gel
electrophoresis, check insert size
* (and Sanger sequence)
Molecular cloning
Engineering with REs can be difficult
* need restriction sites in the right place
* need very highly purified enzymes (can be expensive)
=> use PCR
Advantages:
* can isolate and fuse fragments independently of restriction sites
Disadvantages:
* length constraints, error-rate of polymerase, GC content
* difficult to join more than one gene/region of interest
To insert more than one gene in a vector, or to join multiple fragments (join fluorescent proteins)
(e.g. gene of interest + tag), there are other methods.
Name a different type of cloning method
TA cloning
taq polymerase is cheap and adds overhanging sequence to be made with dNTP to create two overhanging A so cloning will be very efficient, need to buy vector that has the T convenient but expensive.
TOPO TA cloning
TOPOisomerase is an enzyme which cuts one of the two strands, releases torsion in DNA and then the DNA re-aneals.
Expensive but easiest way of cloning in the lab
there aren’t many restriction sites as all you need is TOPO restriction site however there are still other restriction sites even if its expensive to maintain them, this is so that you can cut this site from this plasmid later on.
so buy primers one then they last a very long time
Advantages and disadvantages of some
cloning methods
- Blunt cloning: PCR product and vector with blunt ends. Less efficient than ‘sticky ends’ cloning, non-directional cloning.
- Sticky ends cloning (e.g. with restriction enzymes): incorporate specific restriction sites into primers, PCR amplify the insert → the PCR product will have sticky ends → efficient cloning. Use different restriction sites for directional cloning.
- ‘TA cloning’: Taq DNA polymerase has a terminal transferase activity →adds a non-template A residue to the 3’ end of a PCR product → Taq can be used to add a 3’ A residue to blunt PCR products generated with a high fidelity DNA polymerases. The PCR products with the “sticky” 3’ A residue can be cloned into a commercially available vector with a complementary T overhang. Efficient but non-directional.
What are the other methods for molecular cloning
TA cloning
Gateway cloning
Gibson assembly
Gibson assembly
All reaction happen in one tube at one temperate
the difficult part is designing the correct primers
T5 exonuclease that cuts 5’ to 3’ direction
the polymerase add nucleotides to overhanging regions
Ligase to join regions
Different pieces of DNA at a certain temperature to get a molecule.
Gibson assembly
how does it works ?
- Fragmentation: The DNA fragments to be assembled are typically obtained by PCR or enzymatic digestion.
- End preparation: The fragments are treated with a 5’ exonuclease, which chews back the 5’ ends of the DNA strands, creating single-stranded overhangs. This exonuclease activity is usually provided by a proofreading DNA polymerase with 3’ to 5’ exonuclease activity.
- Annealing: The single-stranded overhangs of the DNA fragments are designed to be complementary to each other, allowing them to anneal. The fragments can then hybridize to each other through base pairing.
- Extension: A DNA polymerase is used to fill in the gaps and synthesize the complementary strands, resulting in the formation of a continuous DNA molecule.
- Repair and Ligation: The gaps between the fragments are sealed using DNA ligase, and any nicks or discontinuities in the backbone are repaired by the cellular repair machinery.
The key innovation of Gibson Assembly is that it enables the assembly of multiple fragments in a single isothermal reaction,
What are the advantages and disadvantages of Gibson assembly?
Advantages:
* seamless joining of any DNA fragments
* no need for REs
* multiple fragments can be joined in one step
* cheaper than de novo synthesis
Limitations
* few fragments at a time
* size limit imposed by PCR (PCR-production of fragments to join
you don’t have scar bits that are disturbing your sequence
What do you do with your cloned gene(s)?
- produce a recombinant protein
- for localisation studies (e.g. gene of interest + GFP,
expression reporter gene) - for in vitro biochemical essay
- recombinant animals for medical research
- study interacting proteins
- analyse the effects of reduced expression levels
