Self study assignment: More recent and convenient cloning systems
Prof. Van Camp
Fundamentals of gene and genome technology
Gene and genome technology
Learning aids:
Several video’s and texts on Gateway cloning and Gibson assembly are available on Blackboard
Aims:
1) Gateway cloning
The student can describe the CcdA/CcdB selection system in cloning with technical details
The student can describe the Gateway cloning system with technical details and required
components
The student can describe the advantages and disadvantages of the Gateway cloning system
The student can list and explain the differences between Gateway cloning and traditional
cloning using restriction enzymes and ligase
The student can design a cloning experiment using the Gateway system (the student knows
which att sites to use in which stage of the cloning experiment)
2) Gibson assembly
The student can describe the Gibson assembly with technical details and required compenents
The student can describe the advantages and disadvantage of Gibson assembly
The student can incorporate Gibson assembly in the theoretical design of cloning
experiments to replace restriction enzyme/ligation steps
, Gateway cloning filmpje
Gateway recombination = a form of site-specific recombination. Very useful if you want to
do different experiments of the same gene in a different vector: switch vectors.
- Homologues recombination: non-specific way to repair any broken DNA-end
- Site-specific recombination: only occurs at very specific DNA sites
Integrase forms a complex with 2 target sites. 4 protein molecules involved and the DNA strands are bend
towards each other to allow for easy exchange. The integrase cut & re-ligate DNA while in this formation.
Restriction enzymes vs. Gateway
RE are used in PCR to create linear DNA pieces of the gene of interest and the vector. The two linear pieces
are then rejoined by ligase to reseal the DNA strands into a circular molecule.
In contrast, Gateway recombination starts and ends with 2 circular DNA molecules. The DNA backbones are
cut & religated by the same enzyme (integrase), while in complex with the DNA. Never linear intermediates
Target sequences for Gateway recombination:
- All share a common central region (black)
- Unlike RE sites, these are not palindromic: provides directionality
- They always ppair together: B&P and L&R
o Reversal reaction can occur but every enzyme only goes in 1
direction
- LR clonase: goes from L & R sites to add B & P
o To reverse the reaction you need BP clonase
The clonase enzyme cuts the central region asymmetrically, generating transient
sticky ends (similar to RE). After strand swapping, the central regions anneal and
ligate into the new product sites.
Steps of gateway recombination
1st step: rate-limiting step in the recombination: formation of the protein-DNA complex. Each monomer of
enzymes binds to 1 site of 1 recognition site, forming a dimer on each att site. The 3D conformation is
different on the 2 sites pair. The 2 dimers then form a complex together with the 2 DNA strands in close
proximity. The formation of the protein-DNA complex is the 1 st step in the recombinase reaction cycle.
2nd step: serine residue in active site of each monomer attacks the phosphate backbone of the DNA within
the central region, which creates a sticky end of DNA and a covalent bound between the protein and the
DNA backbone. The 3D conformation of the complex changes so the strands swap at the sticky ends
3rd step: the enzyme relegates the backbones together. The DNA is now no longer attached to the enzyme,
is free to leave and repeat the reaction at another site. After strand swapping, there are different strands
attached at each site of the central region then before. This results in different att sites then in step 1.
Prof. Van Camp
Fundamentals of gene and genome technology
Gene and genome technology
Learning aids:
Several video’s and texts on Gateway cloning and Gibson assembly are available on Blackboard
Aims:
1) Gateway cloning
The student can describe the CcdA/CcdB selection system in cloning with technical details
The student can describe the Gateway cloning system with technical details and required
components
The student can describe the advantages and disadvantages of the Gateway cloning system
The student can list and explain the differences between Gateway cloning and traditional
cloning using restriction enzymes and ligase
The student can design a cloning experiment using the Gateway system (the student knows
which att sites to use in which stage of the cloning experiment)
2) Gibson assembly
The student can describe the Gibson assembly with technical details and required compenents
The student can describe the advantages and disadvantage of Gibson assembly
The student can incorporate Gibson assembly in the theoretical design of cloning
experiments to replace restriction enzyme/ligation steps
, Gateway cloning filmpje
Gateway recombination = a form of site-specific recombination. Very useful if you want to
do different experiments of the same gene in a different vector: switch vectors.
- Homologues recombination: non-specific way to repair any broken DNA-end
- Site-specific recombination: only occurs at very specific DNA sites
Integrase forms a complex with 2 target sites. 4 protein molecules involved and the DNA strands are bend
towards each other to allow for easy exchange. The integrase cut & re-ligate DNA while in this formation.
Restriction enzymes vs. Gateway
RE are used in PCR to create linear DNA pieces of the gene of interest and the vector. The two linear pieces
are then rejoined by ligase to reseal the DNA strands into a circular molecule.
In contrast, Gateway recombination starts and ends with 2 circular DNA molecules. The DNA backbones are
cut & religated by the same enzyme (integrase), while in complex with the DNA. Never linear intermediates
Target sequences for Gateway recombination:
- All share a common central region (black)
- Unlike RE sites, these are not palindromic: provides directionality
- They always ppair together: B&P and L&R
o Reversal reaction can occur but every enzyme only goes in 1
direction
- LR clonase: goes from L & R sites to add B & P
o To reverse the reaction you need BP clonase
The clonase enzyme cuts the central region asymmetrically, generating transient
sticky ends (similar to RE). After strand swapping, the central regions anneal and
ligate into the new product sites.
Steps of gateway recombination
1st step: rate-limiting step in the recombination: formation of the protein-DNA complex. Each monomer of
enzymes binds to 1 site of 1 recognition site, forming a dimer on each att site. The 3D conformation is
different on the 2 sites pair. The 2 dimers then form a complex together with the 2 DNA strands in close
proximity. The formation of the protein-DNA complex is the 1 st step in the recombinase reaction cycle.
2nd step: serine residue in active site of each monomer attacks the phosphate backbone of the DNA within
the central region, which creates a sticky end of DNA and a covalent bound between the protein and the
DNA backbone. The 3D conformation of the complex changes so the strands swap at the sticky ends
3rd step: the enzyme relegates the backbones together. The DNA is now no longer attached to the enzyme,
is free to leave and repeat the reaction at another site. After strand swapping, there are different strands
attached at each site of the central region then before. This results in different att sites then in step 1.
