Gateway cloning
Genome technology and applications
Explenation
A technique used to clone a gene of interest into a variety of expression systems
In vitro version of the integration and excision recombination reactions that take
place when lambda phages infects bacteria
In vivo: this reaction is done by recombination of attachment sites from the phage
(attP) and the bacteria (attB) the phage integrates into bacterial genome flanked
by new recombination sites (attL & attR)
attL & attR can recombine excision of phage from bacterial chromosome &
regeneration attP & attB
Gateway vector: contains modified version of the att sites
Gateway Technology relies on 2 reactions
BP Reaction
- Between attB sites (flanking insert) and attP sites (of donor vector)
- Reaction catalyzed by BP clonase enzyme mix
- Generates entery clone containing DNA of interest flanked by attL
- As byproduct: ccdB is excised from donor vector
, = rate limiting step formation of protein-DNA complex
Each single unit of enzyme binds to one side of one recognition site forming dimer on
each att site
The 3D conformation is different on the 2 different sites allows them to properly pair
2 dimers come together forming a complex
2. a serine residue at active side of each monomer attack the P-backbone of the DNA within
the central region results in creation of sticky end of DNA as well as a covalent bond
between protein and DNA backbone at this point the 3D conformation of the complex
changes the stands swap at the sticky ends
3. the enzyme relegates the backbones back together, the DNA is no longer covalent
attached to the enzyme free to leave and repeat reaction
LR reaction
Genome technology and applications
Explenation
A technique used to clone a gene of interest into a variety of expression systems
In vitro version of the integration and excision recombination reactions that take
place when lambda phages infects bacteria
In vivo: this reaction is done by recombination of attachment sites from the phage
(attP) and the bacteria (attB) the phage integrates into bacterial genome flanked
by new recombination sites (attL & attR)
attL & attR can recombine excision of phage from bacterial chromosome &
regeneration attP & attB
Gateway vector: contains modified version of the att sites
Gateway Technology relies on 2 reactions
BP Reaction
- Between attB sites (flanking insert) and attP sites (of donor vector)
- Reaction catalyzed by BP clonase enzyme mix
- Generates entery clone containing DNA of interest flanked by attL
- As byproduct: ccdB is excised from donor vector
, = rate limiting step formation of protein-DNA complex
Each single unit of enzyme binds to one side of one recognition site forming dimer on
each att site
The 3D conformation is different on the 2 different sites allows them to properly pair
2 dimers come together forming a complex
2. a serine residue at active side of each monomer attack the P-backbone of the DNA within
the central region results in creation of sticky end of DNA as well as a covalent bond
between protein and DNA backbone at this point the 3D conformation of the complex
changes the stands swap at the sticky ends
3. the enzyme relegates the backbones back together, the DNA is no longer covalent
attached to the enzyme free to leave and repeat reaction
LR reaction
